Compunds and compositions that cause non-apoptotic cell death and uses thereof

ABSTRACT

The present invention relates to erastin analogs, particularly compounds of formulae I and II, including compounds 1-20, 22-24, 34, and 40. The invention also relates to pharmaceutical compositions containing such analogs and to methods of treating conditions in a mammal with such analogs and pharmaceutical compositions.

CLAIM TO BENEFIT

This application is a continuation-in-part of international applicationno. PCT/US2007/014360 filed Jun. 19, 2007, which internationalapplication is incorporated by reference as if recited in full herein.The international application claims the benefit of U.S. ProvisionalApplication No. 60/814,864, filed Jun. 19, 2006, U.S. ProvisionalApplication No. 60/817,665, filed Jun. 29, 2006, and U.S. ProvisionalApplication No. 60/861,560, filed Nov. 29, 2006, each of which isincorporated by reference as if recited in full herein.

GOVERNMENT FUNDING

The work described herein was funded, in whole or in part, by NationalCancer Institute Grant R01CA097061. The United States government mayhave certain rights in the invention.

FIELD OF THE INVENTION

The invention is directed to, inter alia, genotype-selective anti-tumordrugs that induce cell death by a non-apoptotic mechanism. The inventionis further directed to uses of compounds and compositions to treatpatients in need of such genotype-selective anti-tumor drugs.

BACKGROUND

Molecularly targeted therapeutics represent a promising approach tocancer drug discovery¹; examples include Gleevec (imatinib mesylate)²,and Herceptin (trastuzumab)³. A limitation of this approach is that someoncogenic proteins are not amenable to inhibition with a small molecule.For example, the RAS oncoproteins are implicated in the genesis ofnumerous human tumors, but have been difficult to target effectivelywith small molecules⁴. The first rat sarcoma (RAS) oncogene wasdiscovered as a genetic element from the Harvey and Kirsten rat sarcomaviruses with the ability to immortalize mammalian cells⁴⁵⁻⁴⁷. MutatedRAS oncogenes (i.e. HRAS, NRAS and KRAS) are found in 10-20% of allhuman cancers: KRAS mutations are found in >90% of pancreatic cancers,50% of colon cancers and 25% of lung adenocarcinomas; NRAS mutations arefound in 30% of liver cancers and 15% of melanomas, and HRAS mutationsare found in 10% of kidney and bladder cancers⁴⁸. Mice with aKRAS^(G12V) knock-in allele develop bronchiolo-alveolar adenomas⁴⁹⁻⁵²;mice expressing KRAS^(G12V) and CDK4^(R24C) develop sarcomas andpre-neoplastic lesions of the pancreas⁴⁹.

RAS proteins are guanine-nucleotide-binding proteins with GTPaseactivity and are associated with the plasma membrane. In the GTP-boundform, RAS proteins are mitogenic. Mutation of glycine-12 to other aminoacids (including valine, i.e. RAS^(G12V)) results in an oncogenic allelewith constitutive mitogenic, transforming activity and reduced GTPaseactivity⁵³. Four downstream pathways activated by RAS proteins are (i)the RAF/MEK/ERK pathway, which regulates cell-cycle progression, (ii)the PI3K/PDK/AKT pathway, which regulates cell survival, (iii) theRalGDS pathway, which regulates membrane trafficking and vesicleformation, and (iv) the PLC-gamma/PKC pathway, which regulates Ca⁺⁺signaling^(4, 53, 54). Small molecules that activate the GTPase activityof RAS proteins might also be developed, although such an approach maybe challenging to realize⁴. Alternative approaches, such as inhibitingRAS lipidation and processing, have been pursued^(4, 13).

A complementary strategy involves searching broadly foroncogenic-RAS-selective lethal compounds that kill tumor cells only inthe presence of oncogenic RAS⁵. This genotype-selective approach can beapplied to oncogenes such as those of the RAS gene family, whose geneproducts cannot be easily inhibited by a small molecule drug¹². Suchoncogene-selective compounds may target novel proteins inoncoprotein-linked signaling networks. Compounds reported to displayoncogene-dependent lethality include (1) the rapamycin analog CCI-779 inmyeloma cells lacking PTEN⁶, (2) Gleevec in BCR-ABL-transformed cells⁷,and (3) other less well-characterized compounds⁸⁻¹¹.

Therefore, there remains a need to identify compounds that selectivelytarget and inhibit growth of tumor cells.

SUMMARY OF THE INVENTION

In certain aspects, the invention provides methods for identifying anagent, which induces oxidative cell death in tumor cells, the methodcomprising: determining VDAC level in a tumor cell, contacting the tumorcell with an agent, and determining whether the tumor cell dies viaoxidative cell death, and wherein a tumor cell which dies via oxidativedeath indicates that the agent induces oxidative cell death. In certainembodiments, oxidative cell death is defined by the presence ofoxidative species, and/or decreased protein level of VDAC1, VDAC2, VDAC3or any combination thereof. In other embodiments, oxidative cell deathis defined by the presence of oxidative species, and/or decreasedprotein level of VDAC1, VDAC2, VDAC3 or any combination thereof, and theabsence of any one of a number of molecular markers which are associatedwith cell death mechanisms such as apoptosis, necrosis, autophagy, andso forth. Determining of VDAC level can be done by any suitable knownmethod in the art.

In other aspects, the invention provides methods for identifying anagent, which induces oxidative cell death in tumor cells, the methodcomprising: increasing VDAC level in a cell, contacting the cell with anagent, and determining whether the cell dies via oxidative cell death,wherein a cell which dies via oxidative death indicates that the agentinduces oxidative cell death. In certain embodiments, the methodincreases the expression of VDAC1, VDAC2, VDAC3 or any combinationthereof. In certain embodiments, VDAC expression can be increased by anysuitable method known in the art, including nucleofection with vectorcarrying nucleic acid encoding VDAC, stable transfection with nucleicacid encoding VDAC, treatment with any suitable agent which increasesVDAC expression. In some embodiments, such agents can upregulate VDACexpression by targeting or downregulating inhibitors of VDAC expression.In other embodiments, such agents can target upstream effectors of VDACfunction and expression.

In other aspects, the invention provides a method for identifying anagent which induces oxidative cell death in tumor cells, the methodcomprising: providing a tumor cell, contacting the tumor cell with anagent, and determining whether the tumor cell dies via oxidative celldeath, wherein oxidative cell death is determined by (I) detecting: (i)an increased level of oxidative species in the cell; or (ii) a decreasedlevel of VDAC expression in the cell, and (II) identifying one or moreof: (i) a lack of caspase 3 cleavage or activation; (ii) a lack ofcytochrome C release; (iii) a lack of PARP cleavage or activation; (iv)a lack of Annexin V staining; (v) lack of alterations in chromatinmorphology; (vi) a lack of nuclear DNA laddering; (vii) a lack of TUNELstaining of nuclear DNA; (viii) a lack of depletion of ATP levels.

In certain embodiments, the methods of the present invention can beoptionally performed in the presence or absence of a second agentselected from the group consisting of: inhibitors ofmitochondria-generated oxidative species, iron chelators, andanti-oxidants. In certain embodiments of the methods of the presentinvention, determining cell viability compares viability in the presenceor absence of the second agent, wherein loss of cell viability in theabsence of the second agent is indicative of an agent which inducesoxidative cell death. An agent which induces cell death only in theabsence of inhibitors of mitochondria-generated oxidative species, ironchelators, and anti-oxidants, is indicative of an agent which inducesoxidative cell death.

In certain embodiments, the methods of the present invention furthercomprise determining whether mitochondrial morphology is altered,wherein altered mitochondrial morphology is indicative of an agent whichinduced an oxidative cell death. In certain embodiments, alteredmorphology can be detected when mitochondria are enlarged, or fused.

In other aspects, the present invention provides methods for identifyingan agent which induces oxidative cell death in tumor cells, the methodcomprising: providing isolated mitochondria expressing VDAC protein,wherein VDAC protein is VDAC1, VDAC2, or VDAC3, or any isoform thereof,or any combination thereof; contacting the cellular fraction with anagent; and determining whether the agent alters permeability of theouter mitochondrial membrane, wherein an increase in the permeability ofthe outer mitochondrial membrane is indicative of an agent which inducesa non-apoptotic oxidative cell death. In certain embodiments, theisolated mitochondria are in a cellular fraction comprisingmitochondria. In other embodiments, the isolated mitochondria arepurified mitochondria in a lipid bi-layer. In certain embodiments,determining whether the agent alters permeability of the outermitochondrial membrane is done by measuring the levels NADH transport.In other embodiments, whether the agent alters permeability of the outermitochondrial membrane is done by measuring the levels ATP transport.

In other aspects, the invention provides methods for identifying anagent which induces oxidative cell death in tumor cells, the methodcomprising: providing a tumor cell expressing a fluorescently labeledVDAC protein, wherein the VDAC protein is VDAC1, VDAC2, or VDAC3, or anyisoform thereof, or any combination thereof; contacting the tumor cellwith an agent; determining cell viability, and measuring the fluorescentsignal due to the fluorescently labeled VDAC protein, wherein a decreasein cell viability and a decrease in fluorescence due to thefluorescently labeled VDAC protein is indicative of an agent whichinduces an oxidative cell death.

In other aspects, the invention provides methods for identifying anagent which induces oxidative cell death in tumor cells, the methodcomprising: providing a tumor cell expressing a fluorescently labeledVDAC protein comprising two different fluorescent labels, wherein thelabeled VDAC protein exhibits fluorescent emission at a first and secondwavelength when the channel is open, or a first, second and third (FRET)wavelength when the channel is closed; contacting the tumor cell with anagent; determining cell viability, and measuring the fluorescent signaldue to the fluorescently labeled VDAC protein, wherein a decrease incell viability and a decrease in fluorescence due to FRET in the labeledVDAC protein is indicative of an agent which induces an oxidative celldeath. In certain embodiments, the fluorescently labeled VDAC protein isVDAC1, VDAC2, or VDAC3, or any isoform thereof, or any combinationthereof.

In certain aspects, the present invention provides methods fordetermining susceptibility of a tumor cell to an agent which induces anoxidative cell death, the method comprising: providing a tumor cell anda syngeneic non-tumor cell, and measuring a level of VDAC in the tumorcell and the non-tumor cell, wherein an increase in the level of VDAC inthe tumor cell compared to the VDAC level in the non-tumor cell isindicative of a tumor cell, which is susceptible to an agent thatinduces oxidative cell death. In certain embodiments, VDAC protein levelis measured by any suitable method known in the art. In anotherembodiment, VDAC mRNA level is measured by any suitable method known inthe art. In certain embodiments, VDAC protein is VDAC1, VDAC2 or VDAC3,or any isoform, or any combination thereof.

In another aspect, the present invention provides methods fordetermining susceptibility of a tumor cell to an agent, which inducesnon-apoptotic oxidative cell death, the method comprising: providing atumor cell and a syngeneic non-tumor cell, and determining thephosphorylation level of ERK1/2, wherein the presence of aphosphorylated form of ERK1/2 and/or the presence of an increased levelof a phosphorylated form of ERK1/2 is indicative of a tumor cell whichis susceptible to an agent that induces non-apoptotic cell death. Incertain embodiments, the tumor cell and the syngeneic cell are derivedfrom a subject suffering from a tumor.

In other aspects, the invention provides methods for identifying novelRAS-selective lethal compounds. The invention provides compounds withincreased lethality in oncogenic-RAS-expressing tumor cells. In certainembodiments the compound is erastin. In certain aspects the inventionprovides methods for identifying cellular proteins which interact witherastin. In certain embodiments, a cellular target protein whichinteracts with erastin is a VDAC protein, for example, but not limitedto, VDAC1, 2, or 3. In other aspects, the invention provides a methodfor selectively eliminating tumor cells with activated RAS-RAF-MEK-MAPK,for example, signaling by administering genotype-specific anti-tumorcompounds, such as but not limited to, erastin. In other aspects, theinvention provides that erastin is lethal to tumor cells by a mechanismof non-apoptotic, oxidative cell death. In certain embodiments, theoxidative cell death can be determined by measuring the level ofoxidative species.

In certain aspects, the invention is directed to small-molecule-induced,RAS-RAF-MEK-dependent oxidative cell death involving voltage dependentanion channels. Small molecules with oncogene-selective lethality revealnovel functions of oncoproteins and enable creation of tumor selectivedrugs (Kaelin, W. G. The concept of synthetic lethality in the contextof anticancer therapy. Nat Rev Cancer. 5: 689-98 (2005)). The inventiondescribes the mechanism of action of a novel RAS-RAF-MEK-pathwayselective anti-tumor agent. The agent, herein referred to as erastin wasdiscovered in a screen for small molecules that are preferentiallylethal to oncogenic-RAS-expressing tumor cells. In certain embodiments,the invention provides that erastin exhibits greater lethality in tumorcells harboring oncogenic mutations in HRAS, KRAS or BRAF. Innon-limiting examples, affinity purification and mass spectrometry wereused to identify cellular components, including but not limited toproteins, that interact with erastin. In certain aspects, the inventionprovides that erastin acts through mitochondrial voltage-dependent anionchannels (VDACs). In certain embodiments, erastin causes the appearanceof oxidative species in cells with oncogenic RAS or RAF. In certainembodiments, cells with oncogenic RAS or RAF die through an oxidative,non-apoptotic death mechanism.

In certain embodiments, down regulation of VDAC activity or proteinlevels, a non-limiting example of VDAC down regulation is RNAinterference-mediated knockdown of VDAC2 or VDAC3, caused resistance toerastin. In certain embodiments, VDAC2 and 3 isoforms of VDAC areimplicated in erastin's mechanism of action. In other embodiments,wherein purified mitochondria expressing VDAC3 were used, erastinincreased the permeability of the outer mitochondrial membrane,demonstrating that erastin acts through a gain-of-function mechanism, byopening VDAC2 and VDAC3 channels.

In certain embodiments, a screen of ˜24,000 compounds, identifiederastin, which induces rapid death in engineered tumor cells(BJ-TERT/LT/ST/RAS^(V12) cells (Hahn, W. C. et al. Creation of humantumor cells with defined genetic elements. Nature. 400: 464-8. (1999))with oncogenic HRAS^(V12), but not in isogenic, non-tumorigenic cellslacking oncogenic RAS (BJ-TERT/LT/ST cells). In certain embodiments, theselective cell death was not dependent on the rate of cell division oridiosyncratic to this cell line. In certain embodiments, erastin-treatedcells did not display changes in nuclear morphology. In certainembodiments, imaging by electron microscopy revealed changes inmitochondrial morphology, such as enlargement and fusion ofmitochondria. These mitochondrial morphological changes were notobserved in response to staurosporine, hydrogen peroxide or rapamycin,compounds that induce cell death through apoptosis, necrosis andautophagy, respectively.

In certain aspects, the invention provides methods to identify themechanism of erastin's action. Such methods can include, but are notlimited to, methods involving a chemical suppressor screen to identifyknown compounds that prevent erastin-induced cell death. Other methodscan include an affinity purification approach to identify direct targetsof erastin. A suppressor screen using a library of ˜2,000 biologicallyactive compounds (Root, D. E., Flaherty, S. P., Kelley, B. P. &Stockwell, B. R. Biological mechanism profiling using an annotatedcompound library. Chem. Biol. 10: 881-92 (2003)) identifiedantioxidants, including but not limited to alpha-tocopherol, butylatedhydroxytoluene and beta-carotene, as a class of compounds which preventerastin-induced death.

In certain aspects, the invention provides that oxidizing species areformed and detected in response to erastin treatment. In certainembodiments, oxidizing species are detected usingdihydrodichlorofluorescein in BJ-TERT/LT/ST/RAS^(V12) cells, but not inisogenic BJ-TERT cells. In certain embodiments, there is a modestlyincreased sensitivity to erastin in the presence of the Small Toncoprotein (ST), perhaps because ST moderately activates theRAS-RAF-MEK-MAPK pathway. (Frost, J. A. et al. Simian virus 40 small tantigen cooperates with mitogen-activated kinases to stimulate AP-1activity. Mol Cell Biol 14, 6244-52 (1994).) In certain embodiments, theBJ-TERT cell line was used as a comparison cell line because it lacksboth oncogenic HRAS and ST. In certain embodiments, iron chelators, forexample but not limited to desferrioxamine, any one of the compoundspresented in FIG. 30, suppress erastin-induced lethality via oxidativecell death. Suppression of erastin-induced lethality by an ironchelator, suggests that iron-based Fenton chemistry is involved in thiserastin-induced oxidative death. In certain aspects, the inventionprovides that tumor cells other than BJ-TERT/LT/ST/RAS^(V12) die throughthis oxidative mechanism. In certain embodiments, erastin induces celldeath in HT1080 fibrosarcoma cells. In certain embodiments,erastin-induced cell death in HT1080 cells is suppressed byanti-oxidants.

In certain aspects, the invention provides that the oxidizing speciesgenerated in the presence of erastin does not cause PARP1 cleavage,caspase-3 cleavage or cytochrome c release, all hallmarks of apoptosis,indicating this oxidative death is distinct from the oxidative speciesthat appear during some forms of apoptosis due to loss of mitochondrialmembrane potential. Indeed, loss of mitochondrial membrane potentialonly occurred when the cells had died, after 13 hours of erastintreatment.

In certain aspects, the invention provides that erastin does not induceother hallmarks of apoptosis, which is a stereotypical form ofprogrammed cell death activated by many anti-tumor agents (Dolma, S.,Lessnick, S. L., Hahn, W. C. & Stockwell, B. R. Identification ofgenotype-selective antitumor agents using synthetic lethal chemicalscreening in engineered human tumor cells. Cancer Cell. 3: 285-96(2003); Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis isassociated with endogenous endonuclease activation. Nature. 284: 555-6(1980); Kerr, J. F., Wyllie, A. H. & Currie, A. R. Apoptosis: a basicbiological phenomenon with wide-ranging implications in tissue kinetics.Br J Cancer. 26: 239-57 (1972); Martin, S. J. et al. Earlyredistribution of plasma membrane phosphatidylserine is a generalfeature of apoptosis regardless of the initiating stimulus: inhibitionby overexpression of Bcl-2 and Abl. J Exp Med. 182: 1545-56 (1995);Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. & Horvitz, H. R. The C.elegans cell death gene ced-3 encodes a protein similar to mammalianinterleukin-1 beta-converting enzyme. Cell. 75: 641-52 (1993). Suchhallmarks include caspase-3 cleavage and activation, annexin V staining,alterations in chromatin morphology, poly(ADP ribose)polymerase (PARP)cleavage and cytochrome c release from mitochondria (Song, Z. & Steller,H. Death by design: mechanism and control of apoptosis. Trends CellBiol. 9: M49-52. (1999); Majno, G. & Joris, I. Apoptosis, oncosis, andnecrosis. An overview of cell death. Am J Pathol. 146: 3-15. (1995)).None of these apoptotic markers were activated by erastin. In addition,there is no DNA ladder formation from erastin treated cells, and thereis no suppression of oxidative cell death by pan-caspase inhibitors.Moreover, a classic hallmark of necrotic death, complete depletion ofATP, was not observed in erastin-treated cells. In certain aspects, theinvention provides that erastin induces rapid, oxidative, non-apoptoticdeath in tumor cells with oncogenic HRAS. In other aspects, theinvention provides that tumor cells with activating RAS or RAF mutationsare sensitized to erastin induced cell death. Erastin induced cell deathis not consistent with autophagic cell death or paraptosis. Erastininduced cell death is not suppressed by 3-methyladenine, an inhibitor ofautophagic death. EM analysis further confirmed the absence ofautophagic vesicles. There is no extensive-vacuolation seen as inparaptosis. Erastin induced cell death is non-necrotic. Erastin inducedcell death is suppressed by treatment with cyclohexamide, whichindicates that erastin leads to cell death via an active mechanism.Therefore, erastin induced cell death is an active cell death process,which is different from any of the previously characterized cell deathpathways.

The observation that mitochondrial morphology was perturbed upon erastintreatment suggested that erastin-induced oxidative species originate inmitochondria. In certain embodiments, the invention provides that agentswhich inhibit formation of mitochondria-generated oxidative speciessuppress erastin-induced cell death. In certain embodiments, antimycin,a mitochondrial complex III inhibitor (Ho, S. H., Das Gupta, U. &Rieske, J. S. Detection of antimycin-binding subunits of complex III byphotoaffinity-labeling with an azido derivative of antimycin. J BioenergBiomembr. 17: 269-82 (1985); G, V. O. N. J. & Bohrer, C. Inhibition ofelectron transfer from ferrocytochrome b to ubiquinone, cytochrome c1and duroquinone by antimycin. Biochim Biophys Acta. 387: 409-24 (1975)),and 2-methoxyestradiol, a superoxide dismutase inhibitor (Huang, P.,Feng, L., Oldham, E. A., Keating, M. J. & Plunkett, W. Superoxidedismutase as a target for the selective killing of cancer cells. Nature.407: 390-5 (2000); Wood, L. et al. Inhibition of superoxide dismutase by2-methoxyoestradiol analogues and oestrogen derivatives:structure-activity relationships. Anticancer Drug Des. 16: 209-15(2001)), both partially suppress erastin-induced cell death. Bothcompounds act upstream of mitochondria-generated hydrogen peroxide andhydroxyl radical (potential oxidative species). However, melatonin, aperoxynitrite scavenger (Gilad, E., Cuzzocrea, S., Zingarelli, B.,Salzman, A. L. & Szabo, C. Melatonin is a scavenger of peroxynitrite.Life Sci. 60: PL169-74 (1997)), did not affect erastin-induced celldeath, suggesting peroxynitrite is not involved. Therefore,erastin-induced cell death involves oxidative species emanating frommitochondria. Furthermore, gating of mitochondrial outer membranepermeability is a physiologically important process.

In other aspects, the invention provides that mitochondria, and notperoxisomes, are the source of erastin-induced oxidative species. Incertain embodiments, peroxisome proliferators, for example,ciprofibrate, ciglitazone and clofibrate, and xanthine oxidaseinhibitors, for example oxypurinol and allopurinol, did not affecterastin-induced cell death. In other embodiments, lipoxygenaseinhibitors, prostaglandins, arachidonate esters and acids, andthromboxane receptor antagonists had no effect on erastin-induced celldeath, suggesting lipoxygenases and arachidonic acid pathways are notinvolved. In addition, verapamil, which is a multidrug resistance (MDR)pump inhibitor had no effect on erastin sensitivity, suggesting MDRactivity is not involved in the differential sensitivity of cells toerastin. Thus, erastin induces mitochondrial dysfunction in oncogenicRAS-expressing cells, wherein this mitochondrial dysfunction does notresult in apoptosis or energy failure.

Erastin was discovered, e.g., in a screen for oncogenic-HRAS-selectivelethal compounds. KRAS is more frequently mutated in human cancers thanHRAS. Thus, whether erastin is selectively lethal to tumor cellsharboring oncogenic KRAS was tested. Calu-1 is a lung carcinoma cellline (Calu-1) with an activating mutation in KRAS. In certainembodiments, Calu-1 cells were sensitive to erastin (IC₅₀=5 μM). Twodifferent lentiviral constructs were used to reduce expression of mutantKRAS. Reduction of mutant KRAS levels leads to significant resistance toerastin.

In certain aspects, the invention provides that erastin is lethal totumor cells with activating mutations in proteins downstream of RASproteins. Dose-response of 30 tumor cell lines to erastin was measured,and at least 50% inhibition of cell viability in 19 of the 30 tumor celllines was formed. Numerous sarcoma-derived tumor cell lines weresensitive to erastin, consistent with the fact that erastin wasdiscovered in an engineered tumor cell line created from humanfibroblasts. Non-limiting examples include, HT1080 fibrosarcoma cells.HT1080 fibrosarcoma cells, which have a known activating mutation inNRAS, (Plattner, R. et al. Differential contribution of the ERK and JNKmitogen-activated protein kinase cascades to Ras transformation ofHT1080 fibrosarcoma and DLD-1 colon carcinoma cells. Oncogene. 18:1807-17 (1999)), were quite sensitive to erastin. In order to determinewhether RAS-activated signaling was modulated in cell lines that respondto erastin, the phosphorylation status of ERK1/2 in 12 sarcoma celllines with a range of sensitivities to erastin was evaluated. In certainaspects, the invention provides a non-zero correlation, such as forexample, a correlation coefficient of 0.41, between ERK1/2phosphorylation status and erastin sensitivity in these cell lines. Thisindicates that although ERK1/2 phosphorylation does not directly predictsensitivity to erastin, it may, in some cases, be a proxy for erastinsensitivity.

In certain aspects the invention provides that erastin acts in a mannerthat is specific to cells with activated RAS-RAF-MAPK pathway signaling.A non-limiting example of a cell line with moderate sensitivity toerastin is A673, which has an activating V600E mutation in BRAF, whichis a direct target of RAS proteins (Davies, H. et al. Mutations of theBRAF gene in human cancer. Nature. 417: 949-54 (2002)). To determinewhether the activating mutation in BRAF influences erastin sensitivity,short hairpin RNA-expressing plasmids targeting either BRAF mRNA or, asa control, luciferase (LUC) mRNA were created. Knockdown cell linescontaining these constructs were generated, and their sensitivity toerastin was measured. In certain embodiments, the A673 cells containingeither of these two different BRAF-targeted shRNAs were resistant toerastin. Knockdown of BRAF was confirmed at the protein level by westernblot. In other embodiments, co-expression of a non-targetable V600Emutant BRAF could partially restore sensitivity of these cells toerastin.

In certain aspects, the invention provides that activated RAS-RAF-MEKsignaling renders tumor cells sensitive to erastin. In certainembodiments, treatment of tumor cells with different MEK1/2 inhibitors,which block MEK1/2 signaling, lead to loss of erastin sensitivity intumor cells. In certain embodiments, three different inhibitors causederastin resistance in both BJ-TERT/LT/ST/RAS^(V12) and HT1080 cells,with activating mutations in HRAS and NRAS, respectively. In certainaspects, the invention provides that erastin selectivity kills cells inwhich the RAS-RAF-MEK pathway is constitutively activated.

The invention further provides methods to define erastin's mechanism ofaction. A non-limiting example is a method for affinity-based targetidentification. In certain embodiments of this method, erastin analogsthat could be linked to solid-phase resin for biochemical purificationwere synthesized. A non-limiting example is an erastin-related compound,erastin B1 (compound 53), with the ability to killBJ-TERT/LT/ST/RAS^(V12) fibroblasts expressing oncogenic HRAS, but notisogenic BJ-TERT cells lacking oncogenic HRAS and ST (IC₅₀=10 μM).Replacement of the p-chloro substituent in erastin (compound 36),henceforth referred to as erastin A1 (compound 36), with an aminomethylgroup resulted in an analog (erastin A3 (compound 55)) that retained theability to kill BJ-TERT/LT/ST/RAS^(V12) cells but not BJ-TERT cells.Replacement of the p-fluoro group in erastin B1 with an aminomethylgroup resulted in an analog, referred to erastin B2 (compound 54), thatlacks activity.

Erastin A6 (compound 57), Erastin A3 (compound 55), and erastin B2(compound 54) were immobilized on solid-phase resins, and proteins thatinteract with A3 or A6, but not B2 were identified. In certainembodiments, wherein BJ-TERT/LT/ST/RAS^(V12) cell lysates were used, allthree isoforms of the human mitochondrial voltage-dependent anionchannels (VDAC1, VDAC2 and VDAC3) were identified on the A3 and A6resins, but only VDAC1 on the B2 resin. In certain embodiments, whereinBJ-TERT cell lysates were used, a small amount of VDAC1 on the A3 and A6resins was identified, but none of the VDACs were bound on the B2 resin.Thus, it appears that erastin A3 and A6 more efficiently isolate VDAC2and VDAC3 compared to erastin B2. All three VDACs are identified fromthe pull-downs performed on BJ-TERT/LT/ST/RAS^(V12) cell lysate, whichindicates that VDACs are expressed at a higher level inBJ-TERT/LT/ST/RAS^(V12) cells compared to BJ-TERT cells. Moreover, allthree VDACs are identified with higher confidence from the pull-downsperformed on BJ-TERT/LT/ST/RAS^(V12) cell lysate with erastin A6compared to the pulldowns on BJ-TERT cells with erastin A6, suggestingVDACs are more readily purified from BJ-TERT/LT/ST/RAS^(V12) cellscompared to BJ-TERT cells.

VDACs, also known as eukaryotic porins, are membrane-spanning channelsthat facilitate transmembrane transport of ions and metabolites (Graham,B. H. & Craigen, W. J. Genetic approaches to analyzing mitochondrialouter membrane permeability. Curr Top Dev Biol. 59: 87-118 (2004);Baker, M. A., Lane, D. J., Ly, J. D., De Pinto, V. & Lawen, A. VDAC1 isa transplasma membrane NADH-ferricyanide reductase J Biol Chem. 279:4811-9 (2004)), most notably across the outer mitochondrial membrane(Rostovtseva, T. & Colombini, M. ATP flux is controlled by avoltage-gated channel from the mitochondrial outer membrane. J BiolChem. 271: 28006-8 (1996)). There are three human VDAC genes, VDAC1,VDAC2 and VDAC3, of which VDAC1 is the most studied (Graham et al.,(2004); Rahmani, Z., Maunoury, C. & Siddiqui, A. Isolation of a novelhuman voltage-dependent anion channel gene. Eur J Hum Genet. 6: 337-40(1998)). The three gene products are ˜70% identical and likely havedistinct cellular and organismal functions (Graham et al., (2004)).Although no atomic-resolution structure of VDAC is available, they havebeen proposed to adopt a beta barrel fold analogous to the bacterialporins (Casadio, R., Jacoboni, I., Messina, A. & De Pinto, V. A 3D modelof the voltage-dependent anion channel (VDAC). FEBS Lett. 520:1-7(2002); Forte, M., Guy, H. R. & Mannella, C. A. Molecular genetics ofthe VDAC ion channel: structural model and sequence analysis. J BioenergBiomembr. 19: 341-50 (1987); Shao, L., Kinnally, K. W. & Mannella, C. A.Circular dichroism studies of the mitochondrial channel, VDAC, fromNeurospora crassa. Biophys J. 71: 778-86 (1996)). The finding thaterastin pulls down a mitochondrial protein (VDAC) is consistent with theobservation that erastin induces a mitochondria-driven oxidative death.

In certain aspects, the invention provides that altered expression ofVDACs contributes to erastin sensitivity. To determine whether VDACs areupregulated in response to oncogenic RAS, VDAC abundance was measured,using an antibody that recognizes all three isoforms, in the BJ cellseries (primary BJ cells, BJ-TERT cells containing hTERT, BJ-TERT/LT/STcells containing additionally LT and ST, or BJ-TERT/LT/ST/RAS^(V12)cells containing additionally HRAS^(G12V)). In BJ-TERT/LT/ST/RAS^(V12)cells, total VDAC protein is increased relative to these other celllines.

In other embodiments, after 8 hours of erastin treatment, VDAC3 was nolonger detectable, and after 10 hours, VDAC2 also became undetectable.This type of downregulation has also been observed in the case oftreatment with camptothecin which targets topoisomerase I. Thisindicates that erastin acts by a gain-of-function mechanism and thatcells with more VDAC protein are more sensitive to erastin. A similargain-of-function mechanism occurs with doxorubicin and topoisomerase IIalpha, and with camptothecin and topoisomerase I (Beck, W. T. & Danks,M. K. Mechanisms of resistance to drugs that inhibit DNA topoisomerases.Semin Cancer Biol. 2: 235-44 (1991)) suggesting that a cellular responseto erastin may be the downregulation of VDAC2/3 after lethal oxidativespecies have been generated, as occurs with camptothecin andtopoisomerase I and DNA damage. The fact that VDAC1 is still present atthese later time points suggests that the loss of VDAC2/3 is not simplydue to loss of mitochondria.

To test this gain-of-function hypothesis, VDAC protein levels werereduced using a lentiviral shRNA expression system (Moffat et al. 2006).Five shRNA constructs were created targeting each VDAC isoform and theireffects on erastin resistance were tested. In certain embodiments,knockdown of VDAC3 caused significant resistance to erastin. In anotherembodiment, there is a degree of erastin resistance when VDAC2 wasknocked down. The isoform specificity of each shRNA reagent wasconfirmed at the mRNA and protein level. These results are consistentwith a gain of function mechanism, such as increasing permeability ofthe outer mitochondrial membrane. In contrast, overexpression of VDAC3alone in BJ-TERT cells yielded no increase in sensitivity to erastin,suggesting that other downstream aspects of RAS-RAF-MEK signaling areneeded to sensitize cells to erastin, such as increasing rates ofglycolysis and respiration. Overall, these results are consistent with again of function mechanism involving erastin and VDAC2/3. This effect isspecific to erastin, but not other lethal compounds, e.g.,VDAC2-deficient embryonic stem cells have been shown to be moresensitive, not less sensitive, to staurosporine and etoposide. (Cheng,E. H., Sheiko, T. V., Fisher, J. K., Craigen, W. J. & Korsmeyer, S. J.VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 301,513-7 (2003).)

In certain embodiments, mitochondria can be purified from yeast that hadbeen engineered to express murine or human VDAC3 in place of yeast VDAC.A previous report demonstrated that the rate of NADH uptake through theouter membrane of such mitochondria is dependent on the specific VDACexpressed in yeast, such as for example, murine VDAC3 in these yeast. Incertain embodiments, erastin treatment increases the permeability ofmurine or human VDAC3-expressing mitochondria to NADH, consistent withthe proposed gain-of-function mechanism involving channel opening. Inother embodiments, erastin treatment increases the permeability ofmurine or human VDAC3-expressing mitochondria to NADH. Little intrinsicmembrane permeability was found with VDAC3, consistent with previousreports that VDAC3 does not gate well in vitro. (Xu, X., Decker, W.,Sampson, M. J., Craigen, W. J. & Colombini, M. Mouse VDAC isoformsexpressed in yeast: channel properties and their roles in mitochondrialouter membrane permeability. J Membr Biol 170, 89-102 (1999).) Aninactive analog of erastin, erastin A8 (compound 34), had no effect onmitochondrial membrane permeability. These results suggest that erastinaffects VDAC gating, possibly switching their ion selectivity andallowing cationic species into mitochondria.

Given the interactions between erastin and VDAC using affinity-basedtarget identification and VDAC functional assays, the direct binding oferastin to VDACs was investigated. Using modified versions of previouslyreported protocols, VDAC2 was isolated from E. coli for use in acompetition binding experiment using a radioactively labeled analog(erastin A9 (compound 3). (Poyurovsky, M. V. et al. Nucleotide bindingby the Mdm2 RING domain facilitates Arf-independent Mdm2 nucleolarlocalization. Mol Cell 12, 875-87 (2003); Koppel, D. A. et al. Bacterialexpression and characterization of the mitochondrial outer membranechannel. Effects of n-terminal modifications. J Biol Chem 273, 13794-800(1998).) The results demonstrate that the RAS-selective lethal erastinA9 (IC₅₀: 1.9 μM, FIG. 20 n), unlike inactive erastin analog A8,directly binds to VDAC2 (K_(D): 112 nM, FIG. 21 k), in the processcompeting off radiolabeled erastin A9.

In certain aspects, the invention provides that erastin interacts withVDAC proteins to induce mitochondrial dysfunction, release of oxidativespecies and, ultimately, non-apoptotic, oxidative cell death. Thisprocess appears to be selective for cells with activated RAS-RAF-MEKsignaling. In certain aspects, the invention provides methods toidentify oncogene-selective compounds and to use the identifiedoncogene-selective compounds to illuminate oncogene-related cell deathmechanisms.

Unlike bacterial porins, the eukaryotic VDACs are gated by membranevoltage, at least in vitro (Mannella, C. A. Minireview: on the structureand gating mechanism of the mitochondrial channel, VDAC. J BioenergBiomembr. 29: 525-31 (1997)). In the closed state, ions, but not smallmolecule metabolites, can penetrate through VDAC pores (Mannella, C. A.Minireview: on the structure and gating mechanism of the mitochondrialchannel, VDAC. J Bioenerg Biomembr. 29: 525-31 (1997)). In the openstate, both ions and metabolites pass through VDAC channels. Themechanism and frequency of channel gating in vivo is not known, althoughprotein regulators of VDAC gating are reported to exist (Kmita, H.,Budzinska, M. & Stobienia, O. Modulation of the voltage-dependentanion-selective channel by cytoplasmic proteins from wild type and thechannel depleted cells of Saccharomyces cerevisiae. Acta Biochim Pol.50: 415-24 (2003)). VDACs have also been reported to interact with BCLproteins and participate in the formation of the mitochondrialpermeability transition pore that facilitates release of cytochrome cfrom mitochondria (Shimizu, S., Narita, M. & Tsujimoto, Y. Bcl-2 familyproteins regulate the release of apoptogenic cytochrome c by themitochondrial channel VDAC. Nature. 399: 483-7 (1999); Rostovtseva, T.K., Tan, W. & Colombini, M. On the Role of VDAC in Apoptosis: Fact andFiction. J Bioenerg Biomembr. 37: 12942 (2005); Chandra, D., Choy, G.,Daniel, P. T. & Tang, D. G. Bax-dependent regulation of Bak byvoltage-dependent anion channel 2. J Biol. Chem. 280: 19051-61 (2005)).This is a critical event in the intrinsic, mitochondria-driven apoptoticpathway, but does not appear to be involved in erastin's mode of action.

In certain aspects, the invention provides that erastin interacts withVDACs or a VDAC-containing mitochondrial outer membrane complex toinduce mitochondrial dysfunction, such as for example, alteredmitochondrial morphology, changes in the permeability of the outermitochondrial membrane, increased respiration, which can be measured bydetermining oxygen consumption, increased leakage of oxidative species,which can be measured with dihydrodichlorofluorescein or other dyes,release of oxidative species and non-apoptotic cell death. In certainembodiments, oxidative cell death is selective for cells with activatedRAS or RAF signaling. Erastin's effect on tumor cells is likely becauseRAS and RAF proteins upregulate VDACs, and by activating RAF familymembers, which have been reported to inhibit VDACs (Le Mellay, V.,Troppmair, J., Benz, R. & Rapp, U. R. Negative regulation ofmitochondrial VDAC channels by C-Raf kinase. BMC Cell Biol. 3: 14(2002)). Thus, cells with greater RAS/RAF activity are likely to have anincreased pool of latent VDACs and are, therefore, more susceptible tocompounds that disregulate VDAC function. In certain aspects, theinvention provides methods to discover oncogene-selective compounds, andthe use of such compounds to illuminate novel oncogene-specific celldeath mechanisms.

In another embodiment, the invention is an erastin analog, such as acompound having a structure defined by formula I:

whereinR₁ is a hydrophobic or hydrophilic substituent, which is attached to oneor more positions of at least one carbon atom of the ring;R₂ is selected from H, C₁₋₈alkyl, C₁₋₈alkoxy, 3- to 8-memberedcarbocyclic or heterocyclic, aryl, heteroaryl, C₁₋₄aralkyl, residues ofglycolic acid, ethylene glycol/propylene glycol copolymers, carboxylate,ester, amide, carbohydrate, amino acid, alditol, OC(R₆)₂COOH,SC(R₆)₂COOH, NHCHR₆COOH, COR₇, CO₂R₇, sulfate, sulfonamide, sulfoxide,sulfonate, sulfone, thioalkyl, thioester, propylphthalimide, andthioether;R₃ is a C₂₋₈ alkoxy;R₆ is selected from H, C₁₋₈alkyl, carbocycle, aryl, heteroaryl,heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle, whereineach alkyl, carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, and alkylheterocycle may be optionally substituted withat least one substituent;R₇ is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl, aryl,carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, alkylheterocycle, and heteroaromatic may be optionallysubstituted with at least one substituent; oran enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.

In another embodiment, the invention is an erastin analog, such as acompound having a structure defined by formula Ia:

whereinR₁ is a hydrophobic or hydrophilic substituent, which is attached to oneor more positions of at least one carbon atom of the ring;R₃ is a C₂₋₈ alkoxy; oran enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.

In another embodiment, the invention is an erastin analog, such as acompound having a structure defined by formula II:

whereinA is selected from the group consisting of C, N, and O;R₁ is a hydrophobic or hydrophilic substituent, which is attached to oneor more positions of at least one carbon atom of the ring with theproviso that when A is C, R₁ is not NH₂ or NO₂;R₂ is selected from H, C₁₋₈alkyl, C₁₋₈alkoxy, 3- to 8-memberedcarbocyclic or heterocyclic, aryl, heteroaryl, C₁₋₄aralkyl, residues ofglycolic acid, ethylene glycol/propylene glycol copolymers, carboxylate,ester, amide, carbohydrate, amino acid, alditol, OC(R₆)₂COOH,SC(R₆)₂COOH, NHCHR₆COOH, COR₇, CO₂R₇, sulfate, sulfonamide, sulfoxide,sulfonate, sulfone, thioalkyl, thioester, propylphthalimide, andthioether;R₃ is a C₂₋₈ alkoxy;R₄ is a hydrophilic substituent, which is attached to at least oneposition of A, except that when A is O, R₃ is nothing;R₆ is selected from H, C₁₋₈alkyl, carbocycle, aryl, heteroaryl,heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle, whereineach alkyl, carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, and alkylheterocycle may be optionally substituted withat least one substituent;R₇ is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl, aryl,carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, alkylheterocycle, and heteroaromatic may be optionallysubstituted with at least one substituent; oran enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.

In another embodiment, the invention is an erastin analog, such as acompound selected from the group consisting of formula IIa, IIb, andIIc:

whereinR₁ is a hydrophobic or hydrophilic substituent, which is attached to oneor more positions of at least one carbon atom of the ring with theproviso that in formula IIa, R₁ is not NH₂ or NO₂;R₂ is selected from H, C₁₋₈alkyl;R₃ is a C₂₋₈ alkoxy;R₄ and R₅, when present, are independently selected from the groupconsisting of H and an hydrophilic substituent; oran enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.

In another embodiment, the invention is a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound offormula I or II.

In another embodiment, the invention is a method of treating a conditionin a mammal, which comprises administering to the mammal atherapeutically effective amount of a compound or a pharmaceuticalcomposition containing a compound of formula I or II.

In another embodiment, the invention is an erastin analog, such as acompound selected from:

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound selected from:

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.

A further embodiment of the invention is a method of treating acondition in a mammal. This method comprises administering to the mammala therapeutically effective amount of a compound selected from:

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.

Another embodiment of the invention is a method of treating a conditionin a mammal comprising administering to the mammal a therapeuticallyeffective amount of a pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound selected from:

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

The application contains at least one drawing executed in color. Copiesof this patent application publication or any patent to issue therefromwith color drawing(s) will be provided by the Patent and TrademarkOffice upon request and payment of the necessary fee.

FIG. 1 shows the steps to produce experimentally transformed humancells. BJ cells are primary human foreskin fibroblasts. BJ-TERT cellsare derived from BJ cells and express hTERT, the catalytic subunit ofthe enzyme telomerase. BJ-TERT/LT/ST cells are derived from BJ-TERTcells by introduction of a genomic construct encoding both simian virus40 large (LT) and small T (ST) oncoproteins. BJ-TERT/LT/ST/RAS^(V12)tumor cells (also referred to as BJELR cells) are derived fromBJ-TERT/LT/ST cells by introduction of an oncogenic allele of HRAS(RAS^(V12)). BJ-TERT/LT/RAS^(V12) cells are derived from BJ cells byintroduction of cDNA constructs encoding TERT, LT, HRAS^(V12), and acontrol vector. BJ-TERT/LT/RAS^(V12)/ST cells are derived fromBJ-TERT/LT/RAS^(V12) cells by introduction of a cDNA encoding ST.

FIG. 2 shows RAS-selective lethality of erastin in engineered tumorcells. Erastin (compound 36) was tested in a dilution series in fiveengineered cell lines derived from primary BJ fibroblasts. Cells wereincubated with erastin for two days at 37° C. with 5% CO₂, Alamar Bluewas added (10% final volume) and fluorescence measured (530 nm ex, 590nm em). Cell lines harboring oncogenic RAS (triangles, circles and x's)were sensitive to erastin, but isogenic lines lacking oncogenic RAS(squares and diamonds) were resistant. Two different clones ofengineered tumor cells (circles and triangles) responded similarly. Inaddition, replacement of the viral oncogene LT with mutants of p53, CDK4and cyclin D did not change erastin sensitivity (purple X's).

FIG. 3 shows growth rates of engineered cell lines. The relative growthrates of five cell lines were assessed by seeding an equal number ofcells and measuring cell number using Alamar Blue metabolism after 1, 2and 3 days. BJ-TERT/LT/ST/RAS^(V12) cells grow faster than the otherlines. BJ-TERT cells grow more slowly than the other cell lines. Therates of growth of the other three cell lines are the same. Thisinformation was used to ensure that RAS-selective lethal compounds suchas erastin are not simply selective for rapidly dividing cells.Compounds with RAS selectivity should differentially affect the threecell lines that grow at the same rate but have different oncogenic RASstatus.

FIG. 4 shows that oncogenic RAS activates numerous signaling pathways.The RAS-RAF-MEK-ERK pathway is particularly important in causingsensitivity to erastin-induced death. From Malumbres and Barbacid,Nature Reviews Cancer, 3:7-13 (2003).

FIG. 5 shows that PARP1 is not cleaved in response to erastin treatment.BJ-TERT/LT/ST/RAS^(V12) tumor cells were seeded in six-well dishes,incubated overnight at 37° C. with 5% CO₂ and were treated with nothing(NT), staurosporine (ST, 1 μM) for 6 h, camptothecin (C, 1 μM) for 18 h,or erastin (20 μg/mL) for the indicated time, lysed and analyzed bywestern blot with an anti-PARP1 antibody. A relatively highconcentration of erastin was used to ensure loss of PARP cleavage wasnot due to a concentration-dependent effect. PARP1 (top band) is cleavedfrom 110 kD to 85 kD in response to staurosporine, is degraded inresponse to camptothecin, and is unaffected by erastin (by 18 h,erastin-treated cells are almost all dead and little protein remains).

FIG. 6 shows that erastin does not induce cytochrome c release frommitochondria. BJ-TERT/LT/ST/RAS^(V12) cells were seeded in polystyrene100×20 mm dishes in 10 ml media. After overnight incubation at 37° C.with 5% CO₂, the cells were treated with nothing (NT), staurosporine(ST, 1 μM) or erastin (20 μg/mL) for 6 h, washed with 10 mL ice-cold PBSand lysed by passage through a 25-gauge needle (five strokes). Celllysates were centrifuged at 1850 rpm for 5 min at 4° C. to remove thenuclear fraction. Mitochondria were removed from the soluble cytosolicfraction by pelleting at 10,000 rpm. Supernatant and mitochondrialpellets were solubilized in SDS-PAGE loading buffer and analyzed bywestern blot using anti-cytochrome c and pan-VDAC antibodies andIR-dye-linked secondary antibodies. VDACs are mitochondrial proteins, sotheir absence in the cytosolic lane indicates effective separation ofcytosol (C) from mitochondria (M). Fluorescence was detected on a LI-COROdyssey infrared scanner.

FIG. 7 shows that anti-oxidants suppress erastin-induced death.BJ-TERT/LT/ST/RAS^(V12) cells were treated as indicated for 24 hours andphotographed. Abbreviations: DMSO, dimethylsulfoxide; BHT, butylatedhydroxytoluene [erastin]=9 μM.

FIG. 8 shows that erastin-induced formation of oxidative speciesrequires activated RAS signaling. BJ-TERT/LT/ST/RAS^(V12) cells (shownin histogram plots and in line graph, dark line) and BJ-TERT cells(shown in line graph only, light line) were treated with 4.6-μM erastinand the level of intracellular oxidative species determined using2′7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA, Molecular Probes)and flow cytometry. Cells were seeded at 3×10⁵ cells per dish in 60-mmdishes and allowed to grow overnight. Cells were incubated with 10 μM ofH₂DCF-DA for 10 minutes, harvested by trypsinization, washed twice withcold PBS, re-suspended in 100 μl of PBS and incubated with 5 μl of50-μg/ml propidium iodide for 10 minutes. 400 μl of PBS was added andthe solution analyzed by flow cytometry (FACSCalibur-Becton-Dickinson).

FIG. 9 shows that knockdown of KRAS in Calu-1 lung cancer cells causesresistance to erastin. (Left panel) Calu-1 cells that had been infectedwith lentiviruses expressing shRNAs targeting GFP (as a control) or KRAS(targeting 19 nucleotides starting at nucleotide 269 or 509) were seededin 6-well plates, treated with erastin and viability measured usingTrypan Blue exclusion on a Beckman Vi-Cell. Error bars represent one SD.(Right panel) Western blot showing KRAS knockdown in Calu-1 cells. Topband=actin, Bottom band=KRAS. Calu-1 cells were infected with nothing(left lane), or lentiviruses containing shRNA constructs targeting GFPor KRAS. For KRAS, the shRNA was designed to match 19 nucleotidesstarting at the indicated nt.

FIG. 10 shows that VDAC proteins are more abundant in the presence ofoncogenic RAS. Western blot of BJ-derived engineered cell lysates usinga pan-VDAC antibody (Abcam, top band) that recognizes all threeisoforms. A control protein (eIF4E) is shown as a loading control. Theratio of VDAC/eIF4E is plotted for each cell line. Error bars representone standard deviation. Membranes were scanned using the Licor OdysseyImaging System.

FIG. 11 shows that oncogenic BRAF is required for erastin sensitivity.A673 cells and stable derivatives were treated with erastin, andviability measured using Alamar Blue (Panel on the right). The stablelines expressed a short hairpin RNA (shRNA) targeting BRAF or, as acontrol, luciferase. A673 cells were infected with pSIRIPP-derivedretroviruses expressing shRNAs against BRAF or luciferase, and wereselected in 2 μg/ml puromycin. Knockdown was confirmed by western blot(panel on the left)−lane 1 (left): A673 cells, lane 2 (middle):+shRNA(luc), lane 3 (right): +shRNA(BRAF). Top band=BRAF, bottomband=tubulin (loading control).

FIG. 12 shows that MEK1/2 inhibitors suppress erastin lethality.BJ-TERT/LT/ST/RAS^(V12) engineered tumor cells were treated with theindicated concentrations of erastin and each MEK1/2 inhibitor for 24hours and viability was determined by Trypan Blue exclusion. Similarresults were obtained in HT1080 tumor cells harboring an NRAS activatingmutation.

FIG. 13 shows that iron chelators suppress erastin lethality.BJ-TERT/LT/ST/RAS^(V12) cells were treated with erastin and fourdifferent iron chelators, desferrioxamine mesylate (DFOM), the compounds1a and 2a whose structures are shown in FIG. 30, and the compound “311”(311-10 or 311-1) (Green, D. A, et al., Inhibtion of Malignant CellGrowth by 311, a novel iron chelator of the pyridoxal isonicotinoylhydrazone class: Effect on the R2 subunit of ribonulceotide reductase.Clinical Cancer Research, (7): 3574-3579 (2001). Cell killing wasmeasured by Alamar Blue assay.

FIG. 14 shows structures of various erastin analogs of the presentinvention. Erastin A2 (compound 56), lacking the methyl group at R₁, isas active as erastin itself (IC₅₀=2.5 μM). Erastin A3 (compound 55),containing an aminomethyl group at R₂, retains BJELR selective lethality(IC₅₀=25 μM in BJELR cells). Erastin B1 (compound 53) is an activeanalog (IC₅₀=10 μM in BJELR cells). Erastin B2 (compound 54) is aninactive analog of B1 (compound 53), with an aminomethyl group in the R₃position (no lethality up to 50 μM). Thus, erastin A3 was used as anaffinity reagent and erastin B2 was used as a potential negativecontrol, although its loss of activity could be due to other factorsthan target binding, such as membrane permeability

FIG. 15 shows that knockdown of VDAC3 causes resistance to erastin.VDAC3 was knocked down in HT1080 cells using a shRNA vector andviability was measured. FIG. 15A: VDAC3 knockdown was confirmed at theprotein level, 2D gel showing 3 VDAC isoforms and actin. Knockdown wasalso confirmed by qPCR. FIG. 15B: sensitivity to erastin was measured byTrypan Blue exclusion (red line-diamond symbol). As a control, an shRNAtargeting GFP was used (black line-square symbol).

FIG. 16 shows that erastin increases the flux of NADH throughVDAC3-containing outer mitochondrial membranes. Mitochondria wereisolated from yeast expressing murine VDAC3 in place of yeast VDAC. Therate of NADH oxidation by an inner membrane NADH dehydrogenase wasdetermined by measuring the change in NADH absorbance over time.Mitochondria with a disrupted outer membrane show an increased rate ofNADH oxidation (red, shocked) compared to untreated mitochondria(green). Erastin increased the rate of NADH oxidation, presumably byopening VDAC3 (black).

FIG. 17 shows a homology model of VDAC1, bound to erastin. The leftpanel shows a side view of the pore, while the right panel shows atop-down view. Predicted beta strands (yellow) and alpha helices (red).White arrow is pointed at erastin which is docked in a binding site atthe base of the alpha helix. Docking and rendering performed withMolecular Operating Environment (Chemical Computing Group). Coordinates:FEBS Lett. 2002, 5; 520(1-3):1-7.

FIG. 18 shows that VDAC3 knockdown causes resistance to erastin. HT1080fibrosarcoma cells were infected with shRNA targeting VDAC3 andsensitivity to erastin was measured using Trypan Blue exclusion. shRNAto GFP was used as a control (panel A). Knockdown of VDAC3 was confirmedat the protein level by 2D gel and western blot with a pan-VDAC antibody(panels B-D).

FIG. 19 (panels a-m) shows that erastin activates a rapid, oxidative,non-apoptotic cell death process. (a) BJ-TERT/LT/ST/RAS^(V12) cells weretreated with 9 μM erastin A1 for the indicated length of time, and thenumber of viable cells determined. (b) 1,000 BJ-TERT/LT/ST/RAS^(V12)cells/well in 384-well plates were treated with erastin for 2 d, thenAlamar Blue was added to a final concentration of 10% and the plateswere incubated for 16 h. Red fluorescence resulting from reduction ofAlamar Blue was detected on a Victor3 plate reader. The percent growthinhibition is shown; error bars represent one SD. Three differentRAS^(V12)-expressing cell lines were used, and all were found to besensitive to erastin, whereas two isogenic lines lacking RAS^(V12) wereresistant to erastin. (c) TEM images (20,000× magnification) ofBJ-TERT/LT/ST/RAS^(V12) mitochondria after cells were treated withnothing, erastin (37 μM for 10 h) or staurosporine (STS, 1 μM for 5 h),and phase-contrast photograph of BJ-TERT/LT/ST/RAS^(V12) cells 24 hafter 9 μM erastin A1 treatment is also shown to indicate that evenafter cell death, nuclei are intact. (d) Anti-oxidants suppresserastin-induced death. BJ-TERT/LT/ST/RAS^(V12) cells were treated asindicated and photographed. (e) The number of viable cells wasquantified under each condition using a hemacytometer. (f) Ironchelators suppress erastin-induced cell death. Cells were seeded in384-well plates (6,000 cells/well) and treated with 2-fold dilutionseries of erastin in the presence of 100 μM desferrioxamine mesylate(DFOM), 100 μM of iron chelator 1a, 100 μM of iron chelator 2, 10 μM or1 μM of iron chelator 311. After 24 h, Alamar Blue was added and cellviability assayed. (g, h) Erastin-induced formation of oxidativespecies. BJ-TERT/LT/ST/RAS^(V12) (shown in histogram plots, and in linegraph as dark diamonds) or BJ-TERT cells (shown in line graph only,light circles) were treated with 4.6-μM erastin A1 and the level ofintracellular oxidative species determined. (i, j) STS, but not erastin,induces PARP1 cleavage (red bands at top) in BJ-TERT/LT/ST/RAS^(V12)cells and in A673, HT1080 and HeLa cells. NS: non-specific band. (k)Cytochrome c is released from mitochondria in response to STS, but noterastin in BJ-TERT/LT/ST/RAS^(V12) cells. Cells were treated with STS orerastin, separated into mitochondrial and cytosolic fractions, andprobed for cytochrome c (lower red band) and total VDAC (middle greenband, pan-VDAC antibody). (I) Caspase-3 is not cleaved in response toerastin. Cells were treated as indicated and probed with ananti-caspase-3 antibody that recognizes both the full length and thecleaved, active form (lower band). (m) ATP levels are decreased modestlyupon erastin treatment. ATP levels were measured, viable cellsdetermined by Trypan Blue exclusion and ATP per viable cell determined.Abbreviations: DMSO, dimethylsulfoxide; BHT, butylated hydroxytoluene;TOC, alpha-tocopherol; ERA, erastin A1; CAR, beta-carotene; STS,staurosporine.

FIG. 20 (panels a-n) shows that erastin lethality is dependent on theRAS/RAF/MEK pathway. (a) Calu-1 lung carcinoma cells harboring oncogenicKRAS were infected with lentivirus containing shRNAs targeting KRAS orGFP. Sequences of shRNAs are indicated by starting nucleotide (nt) inthe KRAS mRNA coding sequence. Cells were treated with erastin andviability measured by Trypan Blue exclusion. (b) Knockdown of KRAS wasconfirmed by western blot. (c,d) A673 cells harboring oncogenic BRAFwere infected with lentiviral shRNAs targeting BRAF exon 5 (c) or the 5′UTR (d), luciferase (LUC) (c) or GFP (d). Cells were treated witherastin and viability measured using Alamar Blue (c) and Trypan Blue(d). (e) Knockdown of BRAF was confirmed by western blot. (f) MEKinhibitors prevent erastin lethality. BJ-TERT/LT/ST/RAS^(V12) cells weretreated with erastin alone or in with a MEK inhibitor U0126 (20 μM), MEKinhibitor I (10 μM) or MEK 1/2 inhibitor (50 μM). After 48 h, viabilitywas determined using Trypan Blue. (g) Structures of certain erastin andrelated analogs. (h) and (i) Dose-response curves were measured forerastin A3 and erastin B2 in BJ-TERT/LT/ST/RAS^(V12) cells (blacksquares) or BJ-TERT cells (grey circles). (j) Calu-1 lung carcinomacells harboring oncogenic KRAS were infected with lentivirus containingshRNAs targeting KRAS or GFP. Sequences of shRNAs are indicated bystarting nucleotide (nt) in the KRAS mRNA coding sequence. Cells weretreated with erastin and viability (y-axis) measured by Trypan Blueexclusion. Cells were treated with erastin for 24 hours, and percentinhibition of viability (y-axis) measured using Alamar Blue (k) andTrypan Blue (l). (m) Structures of erastin and related analogs. (n)Dose-response curves were measured for erastin A6, erastin B2, erastinA8 and erastin A9 in BJ-TERT/LT/ST/RAS^(V12) cells (squares) or BJ-TERTcells (circles) using Alamar Blue.

FIG. 21 (panels a-k) shows that erastin acts through VDACs. (a)

Quantitative western blot of VDAC/eif4E protein ratio in engineeredBJ-derived cells. (b) Identification of VDAC isoforms inBJ-TERT/LT/ST/RAS^(V12) cells using 2-D electrophoresis. i) VDAC1, VDAC2and VDAC3 were detected by a rabbit polyclonal VDAC antibody(Abcam/ab3434), ii) VDAC1 was detected by a mouse monoclonal VDAC1antibody (Calbiochem/529534), iii) VDAC2 was detected by a goatpolyclonal VDAC2 antibody (Abcam/ab22170), iv) An illustration of 3isoforms of VDAC separated by the 2-D gel electrophoresis. (c)BJ-TERT/LT/ST/RAS^(V12) cells were treated with erastin for theindicated time and levels of each VDAC isoform determined byquantitative 2D western. (d,e) Infection with VDAC3 shRNA protectserastin-treated cells. 293T cells were transfected with shRNA-plasmidconstruct using FuGene and viral supernatant transferred to HT1080cells, and treated with erastin dilutions and viability measured usingTrypan Blue exclusion. Knockdown was confirmed using 2D gels to detectthe 3 VDAC isoforms. VDAC3 shRNAs (V3.B1) or a control construct (shGFP)was used. (f) Quantitative RT-PCR measurements of mRNA levels of the 3VDAC isoforms after infection with shVDAC3. Knockdown was determined bynormalizing to the levels in the shGFP-infected control. Relativeexpression level is shown. Error bars represent one SD. (g) Mitochondriawere purified from yeast expressing murine VDAC3 in place of yeast VDAC,and the rate of NADH uptake determined in the presence or absence oferastin, or in shocked mitochondria in which the outer membrane wasdisrupted. (h) HT1080 cells were infected with virus expressing eitherVDAC3-targeted shRNA-plasmid construct (shVDAC3) or VDAC2-targeted shRNAplasmid (shVDAC2), and knockdown of each VDAC isoform was confirmedusing 2-D protein gels. (i) These cells were then treated with erastindilutions, and viability relative to no treatment (y-axis) wasdetermined using Trypan Blue exclusion and compared to an identicalprocess using a GFP control plasmid. Infection with shVDAC2 protectscells from erastin-induced death. mRNA levels of the three VDAC isoformsafter infection with shVDAC2-expressing virus were measured usingquantitative RT-PCR. (j) Mitochondria were purified from porin-knockoutyeast expressing murine VDAC2, and the rate of NADH oxidation determinedin the presence of erastin or an inactive analog, erastin A8(triangles). Y-axis shows relative rate of NADH oxidation, as normalizedto no treatment. (k) VDAC2 binding assays using tritium-labeled erastinA9 in competition with unlabeled erastin A9 (squares) or erastin A8(triangles) reveals that active analog erastin A9, unlike the inactiveerastin A8, directly binds VDAC2.

FIG. 22 shows that erastin induced death in HT1080 fibrosarcoma cells issuppressed by anti-oxidants.

FIG. 23 shows that knockdown of BRAF by shRNA causes resistance toerastin. Co-expression of a non-targetable V600E mutant BRAF restoressensitivity to erastin.

FIG. 24 shows that inhibitors of MEK protect the viability oferastin-treated BJELR and HT1080 cells. Cells grown in 175 cm² flaskswere reseeded in 6-well format (1.4×10⁵ cells/well) and treated witherastin dilutions (5-fold, from 36 μM to 60 nM, with no-drug control,n=2) in the presence of one of the MEK inhibitors U0126 (20 μM), MEKinhibitor I (10 μM), MEK1/2 inhibitor (50 μM), PD98059 (50 μM), MEKinhibitor II (30 μM), or in the absence of inhibitor. After 48 hours,cells were trypsinized and counted using a Vi-CELL™ Series CellViability Analyzer. Panels (a) and (b) show the dose-response curvesobtained with BJELR and HT1080 cells, respectively, with U0126, MEKinhibitor I, or MEK1/2 inhibitor, or in the absence of inhibitor.Non-linear regression was used to fit curves to the data points usingGraphPad Prism™ software. The bottom of each curve was set to zero.P-values based on comparison between the values fit for the top,logEC₅₀, and the hill slope for the inhibitors versus no inhibitor wereless than 0.0001 for all curves shown. Panel (c) shows the IC₅₀ valuesin μM from the best fit curve of each inhibitor used. It also gives thefold-change in IC₅₀ produced by each inhibitor and the p-value obtainedwhen comparing the curves based only on the logEC₅₀ value.

FIG. 25 (panels A-D) shows the levels of VDAC, tubulin, actin and eIF4Ewere determined in BJ cells, BJEH cells (expressing hTERT), BJEHLT cells(expressing hTERT, and the large and small T oncoproteins from SV40) andBJELR cells (expressing hTERT, and the large and small T oncoproteinsfrom SV40 and oncogenic HRAS). Images were quantified and are plotted asVDAC relative to each control protein. Error bars represent one standarddeviation.

FIG. 26 (panels a-c) shows that transfection with VDAC1 shRNA protectthe viability of erastin-treated HT1080 cells. Briefly, the assay wascarried out as follows: Day 1, 293T cells seeded in 10 cm tissue culturedishes (2×10⁶ cells/dish); Day 2, shRNA-plasmid construct (pLKO.1vector) introduced to cells using FuGene transfection reagent; Day 3,medium changed; Day 4, supernatant transferred to HT1080 cells in 10 cmtissue culture dishes (1×10⁶ cells/dish); Day 5, cells transferred to175 cm² flasks, medium supplemented with puromycin; Days 6 and 7, mediumchanged and supplemented with puromycin; Day 8, samples harvested forWestern Blot and qRT-PCR, or reseeded in 6-well format (5×10⁵cells/well) and treated with erastin dilutions (2-fold, from 10 μM to625 nM, with no-drug control); Day 9, Vicell analysis performed. UniqueVDAC1 shRNAs (V1.161, V1.279, V1.396, V1.607, V1.921), control construct(GFP).

FIG. 27 (panels a-c) shows that transfection with VDAC2 shRNA protectthe viability of erastin-treated HT1080 cells. Briefly, the assay wascarried out as follows: Day 1, 293T cells seeded in 10 cm tissue culturedishes (2×10⁶ cells/dish); Day 2, shRNA-plasmid construct (pLKO.1vector) introduced to cells using FuGene transfection reagent; Day 3,medium changed; Day 4, supernatant transferred to HT1080 cells in 10 cmtissue culture dishes (1×10⁶ cells/dish); Day 5, cells transferred to175 cm² flasks, medium supplemented with puromycin; Day 6, mediumchanged and supplemented with puromycin; Day 7, samples harvested forWestern Blot and qRT-PCR, or reseeded in 6-well format (5×10⁵cells/well) and treated with erastin dilutions (2-fold, from 10 μM to625 nM, with no-drug control). Unique VDAC3 shRNAs (A9, A10, A11, A12),control construct (GFP).

FIG. 28 (panels a-c) shows that transfection with VDAC3 shRNA protectthe viability of erastin-treated HT1080 cells. Briefly, the assay wascarried out as follows: Day 1, 293T cells seeded in 10 cm tissue culturedishes (2×10⁶ cells/dish); Day 2, shRNA-plasmid construct (pLKO.1vector) introduced to cells using FuGene transfection reagent; Day 3,medium changed; Day 4, supernatant transferred to HT1080 cells in 10 cmtissue culture dishes (1×10⁶ cells/dish); Day 5, cells transferred to175 cm² flasks, medium supplemented with puromycin; Days 6 and 7, mediumchanged and supplemented with puromycin; Day 8, samples harvested forWestern Blot and qRT-PCR, or reseeded in 6-well format (5×10⁵cells/well) and treated with erastin dilutions (2-fold, from 10 μM to625 nM, with no-drug control). Unique VDAC3 shRNAs (B1, B2, B4, B6),control construct (GFP).

FIG. 29 (panels a-d) shows representative knockdown experiments usingpLKO.1 shRNA vector. Briefly, HT1080 cells were infected with a virusexpressing a short hairpin RNA (shRNA) to VDAC1, VDAC or VDAC3 and thelevels of each VDAC determined by 2D gel and western blotting with apan-VDAC antibody. The level of eIF4E is shown as a control.

FIG. 30 shows the structures of certain iron chelators used in thepresent invention.

FIG. 31 shows the structures of certain MEK1/2 inhibitors used in thepresent invention.

FIG. 32 (panels a-f) shows that erastin A3 and not its analogue, erastinB2, selectively kills tumor cells; immobilized erastin analogues pulldown different proteins (b); VDAC protein level is increased in RAStumors (c); and VDAC protein level in BJELR cells is increased bynucleofection (d). Quantitative western blot showing the ratio of VDACs(top band, green) to eif4E (bottom band, red) (e). Erastin A1 (erastin),but not podophyllotoxin, is more potent (i.e. effective at a lowerconcentration) in BJELR cells after up regulation of VDACs. Green,untransfected BJELR cells; blue, mock nucleofected BJELR cells; red,VDAC2/3-nucleofected BJELR cells (f).

FIG. 33 shows the activity of aminomethyl substituted erastins. ERA-A6(compound 57), an analog with a p-aminomethyl substituent in place ofthe p-chloro substituent in erastin, is selectively lethal toRAS^(V12)-expressing cells. ERA-B1 (compound 53) is an aminomethylanalog that is inactive and was used as a negative control in pulldownexperiments.

FIG. 34 shows that erastin induces rapid cell death in aRAS^(V12)-dependent fashion. Effect of erastin on Alamar Blue viabilitystaining in BJ-TERT (red) and BJ-TERT/LT/ST/RAS^(V12) (blue) cells.

FIG. 35 shows that erastin potency does not increase with longerexposure. A time-dependent effect of erastin on BJ-TERT andBJ-TERT/LT/ST/RAS^(V12) cells. Cells were seeded in 384-well plates inthe presence of the indicated concentrations of erastin. Inhibition ofcell viability was determined after 24, 48, and 72 hr using calcein AM.

FIG. 36 shows that knockdown of BRAF causes resistance to erastin. A673Ewing sarcoma cells were infected with a lentivirus encoding an shRNA toBRAF or GFP as a control, or parental uninfected cells. Sensitivity toerastin was measured using Trypan Blue exclusion.

FIG. 37 shows mammalian cell death phenotypes. Mammalian cells diethrough several different known mechanisms, including apoptosis,necrosis, mitotic catastrophe, autophagic cell death, paraptosis, or theless well defined methods of dark cell death or oncosis.

FIG. 38 shows that camptothecin, but not erastin, inducescharacteristics of apoptosis. Camptothecin-treated, but noterastin-treated, BJ-TERT/LT/ST/RAS^(V12) cells displayed fragmentednuclei (10%-20% of total nuclei, arrows) as shown. DNA was stained usedHoechst 33342.

FIG. 39 shows that camptothecin-treated, but not erastin-treated,BJ-TERT/LT/ST/RAS^(V12) cells display Annexin V staining. The percentageof cells in the indicated Ml region were 6%, 6%, and 38% in untreated,erastin-treated (9 μM), and camptothecin-treated (1 μM), respectively.

FIG. 40 shows that screens for small molecule suppressors of erastin canreveal the mode of death.

FIG. 41 is a summary of the results from an experiment using flowcytometric analysis using dihydrodichlorofluorescein, which shows thaterastin causes the formation of oxidative species. Briefly, cells weretreated with erastin for the indicated periods of time and oxidativespecies determined using dihydrodichlorofluorescein, which becomes morefluorescent upon oxidation. Hydrogen peroxide treatment was used as acontrol.

FIG. 42 shows a model for the mechanism of erastin-induced cell death.RAS-RAF-MEK signaling causes increased glycolysis and increased VDACexpression. Erastin locks VDACs open and causes dysregulatedrespiration, resulting in oxidative species that react with iron,causing lethal reactive species such as hydroxyl radical.

FIG. 43 shows that over expression of VDAC3 in BJ-TERT cells causes nochange in sensitivity to erastin. (a) BJ-TERT cells, infected with virusexpressing VDAC3 cDNA were treated with erastin dilutions, andviability, relative to no treatment, was measured using Alamar Blueviability analysis. (b) 2D Western Blot analysis indicates a>8-foldincrease in VDAC3 protein expression (arrows) in infected cell linescompared to the parental cell line (BJ-TERT). Unique clones:BJ-TERT.V3.1 and BJ-TERT.V3.2.

FIG. 44 shows the rate of NADH oxidation in mitochondria in the presenceof erastin or an inactive analog, erastin A8. Mitochondria were purifiedfrom yeast expressing murine (a) VDAC1 or (b) VDAC3 in place of yeastVDAC (porin). Y-axes shows rate of NADH oxidation relative to no drugtreatment.

FIG. 45 shows the viability of BJ-TERT/LT/ST/RAS^(V12) cells in responseto 24 hour erastin or erastin B1 analog treatment using an Alamar Blueassay.

FIG. 46 shows the structure and characteristics of certain compoundsaccording to the present invention.

FIG. 47 shows the synthesis of erastin.

FIG. 48 shows the synthesis of certain erastin analogs, includingCompounds 40 and 44, according to the present invention.

FIG. 49 shows the synthesis of additional erastin analogs, includingCompounds 47 and 49, according to the present invention.

FIG. 50 shows the synthesis of additional erastin analogs, includingCompound 2, according to the present invention.

FIG. 51 (panels A-K) shows the effects of various erastin analogsaccording to the present invention in a cell viability assay.

DETAILED DESCRIPTION Definitions

“Oxidative cell death” is a term which refers to cell death which ischaracterized by the increased level of oxidative species measured in acell, altered mitochondrial morphology, including enlarged and/or fusedmitochondria, in the absence of significant increase in mitochondrialnumbers. Oxidative cell death does not manifest typical cellular andmolecular markers of apoptosis, autophagy and/or necrosis.

The term “VDAC” refers to one or more VDAC proteins such as VDAC1,VDAC2, and VDAC3, or any isoform or combination thereof.

BJ-TERT/LT/ST/RAS^(V12) cells are also referred to as BJELR.BJ-TERT/LT/ST cells are also referred to as BJEHLT. BJ-TERT cells arealso referred to as BJEH.

In certain aspects, the invention provides methods to identifygenotype-selective compounds, as well as, compounds and mechanisms thatcause oncogene-selective lethality. Such compounds eliminate tumor cellsharboring specific oncogenic mutations, but have minimal effects onnormal cells lacking these mutations. Small molecules with suchselective lethality reveal functions of oncogenes, and the molecular andcellular pathways affected by oncogenes, and allow for the creation ofselective drugs, which are targeted to specific targets of an oncogenicpathway. In certain aspects, the invention describes a mechanism forselectively eliminating tumor cells, which express oncogenic RASproteins.

An annotated library of biologically active compounds was assembled¹¹⁴.

To identify tumor-selective cytotoxic drugs, including compounds fromthe annotated library, and to study the global patterns of drugactivity, software tools were developed to improve the facility withwhich new tumor-selective compounds can be identified^(114, 120-124).This software was used to discover genotype-selective lethal compoundssuch as erastin⁸. A cheminformatics and laboratory management system forchemical genetic screens was developed as a custom data analysis toolfor analyzing our screening data and we used this to identify erastinand the other RSLs. High-throughput assays generate large quantities ofdata that require sophisticated data analysis tools¹²¹. The softwaretool, SLIMS (Small Laboratory Information Management System), wascreated to facilitate the collection and analysis of large-scalechemical screening data¹²⁴. Compound structures and raw data are loadedinto SLIMS directly from structure data (SD) or plate reader (csv)files; systematic spatial errors can be automatically identified andcorrected using a discrete-Fourier-transformation tool¹²⁰. Publishedliterature associated with active compounds can be automaticallyretrieved from Medline and processed to yield potential mechanisms ofactions¹²¹. This software is available through the website Sourceforge(slims.sourceforge.net).

A gene expression signature-based, high-throughput screening method wascreated in which a gene expression signature is used as a surrogate forcellular states¹¹⁷. The annotated compound library was used to identifycompounds that induce the differentiation of acute myeloid leukemia(AML) cells. The AML gene signature and a differentiated neutrophil genesignature were defined, and multiplexed single base extension massspectrometry (SBE-MS)-based RT-PCR was used to detect this genesignature in 384-well plate format. In screening 1,739 biologicallyactive compounds, 8 compounds were identified that reliably induced thedifferentiation signature and yielded functional evidence ofdifferentiation of AML tumor cells. These results indicate that geneexpression signature-based screening may be useful for chemicalscreening.

A protein-pathway-and-network-alignment software tool was developed. Astargets of compounds that mediate RAS-selective killing are discovered,there is a need for software tools to place these proteins in networksand to identify candidate functions of these proteins. Thenetwork-alignment software tool implements a strategy for aligningprotein-protein interaction networks and pathways that combinesinteraction topology and protein-sequence similarity to identifyconserved protein-interaction pathways and protein complexes¹²³.

To create an annotated library of biologically active compounds,thousands of small molecules with experimentally verified biologicalmechanisms and activities were identified, collected and assembled intoa screenable format¹¹⁴. This annotated library can be used to aid indefining the mechanism of action for RAS-selective lethal compounds,using suppressor and enhancer screens. The library has extensiveannotation to identify, in an unbiased fashion, mechanisms that arestatistically overrepresented among active compounds from a screenversus the parent library¹¹⁴. This approach was used to determine thaterastin acts through an oxidative, non-apoptotic mechanism of celldeath.

Using high-throughput screening of compounds in isogenic, engineeredtumor cell lines, compounds can be discovered that are selectivelylethal to oncogenic-RAS-expressing cells. In certain aspects, theinvention is directed to a compound called erastin that displaysselectivity for tumor cells with activated RAS-RAF signaling. Erastinacts through mitochondrial VDACs to cause an oxidative, non-apoptoticcell death. Defining the mechanism governing erastin-induced cell deathillustrate a means of selectively eliminating tumor cells. In certainaspects, the invention establishes the utility of the genotype-selectivescreening paradigm for discovering anti-tumor agents, including but notlimited to agents that target components of the RAS pathway. In otheraspects, the invention provides VDAC1, 2, and 3 proteins as drug targetsfor anti-cancer agents. Using the tools of synthetic chemistry,molecular biology and proteomics, the invention provides that voltagedependent anion channels (VDACs), including VDAC1, 2, and 3, are targetproteins for one of these compounds, which is named erastin.

The genetic and mechanistic bases of specific drugs' tumor selectivitywere identified through biochemical and molecular approaches. Thegenetic basis of selectivity for eight known agents and erastin weredetermined⁸. Furthermore, the invention describes molecular targets,including but not limited to VDAC molecules, of erastin. The inventionfurther provides the mechanism of erastin-induced cell death, providesVDAC1, 2, and 3 molecules as erastin targets, and provides use oferastin and erastin analogs in vivo in mice.

In other aspects, the invention defines the mechanism by whichmodulation of VDAC activity leads to RAS-selective lethality. In certainembodiments, the invention provides VDAC proteins, including humanVDAC1, 2, and 3, as erastin targets. In other embodiments, the inventionprovides the downstream components and consequences of oncogenic RASsignaling that lead to erastin sensitivity. In other embodiments, theinvention provides the use of optimized erastin analogs in mouse cancermodels.

In certain aspects, the invention provides that erastin acts through themitochondrial VDAC proteins to cause an oxidative, non-apoptotic death.The sensitivity of tumor cells to erastin thus reveals that oncogenicRAS signaling causes increases in VDAC levels and that VDACs aregain-of-function targets for cancer therapeutics. The results supportthe notion of using small molecules to study oncogene function andsuggest that VDAC ligands are potential chemotherapeutic agents for thetreatment of cancers with activated RAS signaling.

In certain aspects, the invention provides that downstream targets ofRAS enable oncogenic-RAS-selective lethality. Furthermore, the inventionprovides a compound, erastin, and some of its cellular targets, the VDACproteins. An affinity-based approach was used to identify the targets oferastin.

Non-Limiting Methods to Determine that a Protein is a Target of a SmallMolecule:

Once a binding protein such as VDAC is identified, it can be determinedwhether binding of a compound to the candidate protein is the basis forthe compound's phenotypic activity. There are a number of methods tovalidate candidate targets. Non-limiting examples of such methods are:(i) RNA-interference knockdown, (ii) cDNA-based overexpression, (iii) invitro binding studies, (iv) photo-crosslinking and (v) creating abinding-defective mutant of the target.

In non-limiting examples, RNA-interference-mediated knockdown andcDNA-based overexpression are methods for decreasing and increasing,respectively, the concentration of a protein. For many small molecules,altering the level of the target protein will alter the compoundpotency. For example, decreasing the concentration of tubulin, thetarget of benomyl, causes increased sensitivity to benomyl²⁸. Incontrast, compounds that act via a gain of function have the oppositerelationship with their target proteins: increasing the concentration oftopoisomerase I, the target of camptothecin, which acts via a gain offunction, causes increased sensitivity to camptothecin^(8, 29-39).

In a non-limiting example, in vitro binding studies can determine thebinding parameters associated with a small-molecule-protein interaction.A candidate protein is overexpressed, purified and incubated with a testcompound. Useful parameters extracted from such experiments are theon-rate, the off-rate, the equilibrium dissociation constant and theentropic and enthalpic contributions to binding affinity. Two methodsused to measure protein-ligand interactions are surface plasmonresonance and isothermal titration calorimetry.

In a non-limiting example, photo-crosslinking can identify a bindingsite for a small molecule on a protein⁴⁰⁻⁴². A photoactivatable moietyis incorporated into a compound to enable crosslinking to a targetprotein. Benzophenones can be photo-activated with long wavelength (>300nm) light, resulting in less destruction of compounds and proteins.Labeled protein is digested with a protease and labeled peptides areidentified with mass spectrometry. In this way, a specific peptidesequence to which a compound is crosslinked is determined, suggestingbinding sites.

In a non-limiting example, creating a mutant protein of a target thatdoes not bind to the test compound can be a useful method of assessingthe functional relevance of the target-ligand interaction. For example,mutants of the mTOR protein that don't bind its ligand rapamycin wereused to show that mTOR is the cellular target of rapamycin^(43, 44).

Creation of a Genome-Scale Lentiviral shRNA Collection:

A powerful method of illuminating the mechanism of action of novelRAS-selective lethal compounds is to perform a large-scale suppressorscreen with RNA interference reagents that reduce expression of specificmRNAs. Such suppressors might reveal direct targets of compounds, orpathways involved in causing sensitivity to them. Towards this end,150,000 shRNA constructs may be created in a lentiviral vector. 90,000constructs were created, sequenced, and protocols needed to performhigh-throughput screens with the library in lentiviral format weredeveloped¹²⁵.

Target Identification Using Photolabeling with Indoxins, Compounds thatOvercome Drug Resistance:

Synthetic lethal screening was used to identify compounds and mechanismsfor overcoming E6-oncoprotein-mediated drug resistance. The screenidentified compounds that potentiate doxorubicin's lethality inE6-expressing colon cancer cells. Tested compounds were derived from theannotated compound library¹¹⁴, the National Institute of NeurologicalDisorders and Stroke (NINDS) library¹²⁶, and a library of compoundspurchased from Timtec, Interbioscreen and Chembridge, herein referred toas TIC library¹²⁷ The screen identified a group of compounds, that werenamed indoxins, that overcome doxorubicin resistance¹²⁸. Indoxinspotentiate doxorubicin, but not camptothecin or podophyllotoxin,suggesting they act at the level of topoisomerase II abundance. It wasfound that indoxins upregulate topoisomerase IIα. When the acylfunctionality was substituted with a biotin-linked group, indoxinsretained activity and selectivity, indicating that affinity reagents canbe introduced at this site. Incorporation of a photo-activatablefunctionality^(129, 130) was achieved by preparing anindoxin-benzophenone-fluorescein photo-reactive probe. Protein targetscross-linked to this probe were purified, eluted and sequenced. Twoproteins were repeatedly pulled-down with the indoxin probe but not acontrol probe: myosin 1C and ARP2¹³¹. The ability of indoxins to targetnuclear myosin 1C could mediate topoisomerase IIα transcriptionalupregulation, as myosin 1C has been linked to transcriptional control:it co-localizes with RNA polymerase II and may affect transcription¹³¹,and is associated with rDNA and required for RNA polymerase IItranscription¹³¹. This demonstrates the photolabeling of a targetprotein using a benzophenone moiety¹²⁸, and that such photolabeling canbe used to identify proteins that interact with the photolabeledcompound.

Discovery of Erastin, a RAS-Selective Lethal Compound:

To discover oncogenic-RAS-selective lethal compounds, an engineeredhuman tumor cell was used (FIG. 1). hTERT, a genomic construct encodingthe Simian Virus 40 large (LT) and small T (ST) oncoproteins, and anoncogenic allele of HRAS (RAS^(V12)) were introduced into primary BJfibroblasts 8, 56, 60. In another series of engineered cells,complementary DNA (cDNA) constructs encoding LT and ST were used inplace of the SV40 genomic construct that encodes both of these viralproteins⁵⁷. In this latter series, ST was introduced in the last stage,enabling the testing of compounds in the presence or absence of ST (FIG.1).

The screen can identify compounds with increased potency and activity inthe presence of RAS^(V12) and/or other genetic elements. 70,000compounds were screened, comprising 20,000 compounds from acombinatorial library, ˜5,000 known biologically active compounds,˜11,000 structurally defined natural products and ˜34,000 drug-likesynthetic compounds¹²⁶⁻¹²⁷. The primary screen tested in triplicate theeffect of treating tumorigenic BJ-TERT/LT/ST/RAS^(V12) cells with eachcompound for 48 hours at a concentration of 4 μg/mL, corresponding to 10μM for a compound with a molecular weight of 400. Also, the screenmeasured cell viability using Alamar Blue, which undergoes a red shiftin fluorescence upon reduction¹³², and calcein AM, which becomesfluorescent when cleaved by intracellular esterases¹³³. Compounds lethalto BJ-TERT/LT/ST/RAS^(V12) cells (>50% inhibition of viability) werere-tested in a two-fold dilution series in isogenic cells with andwithout RAS^(V12), to identify those with RAS^(V12)-dependent lethality.The IC₅₀ value was calculated for each compound in each cell line andthereby five novel compounds were identified that were at leastfour-fold more potent in HRAS^(V12)-expressing cells, compared toHRAS^(V12)-deficient cells⁸.

The engineered tumor cells make use of dominantly acting viraloncoproteins (LT and ST). These viral proteins are involved in celltransformation in specific forms of cancer, namely simian virus40-induced malignant mesothelioma¹³⁴ and other viral oncogenes (E6 andE7) are involved in human papillomavirus-induced cervical carcinoma¹³⁵,and have been used to disrupt p53 and pRB function to transform cells invitro and in vivo¹³⁶⁻¹³⁸. The selectivity of erastin and the othercompounds were further established in a cell line expressing dominantnegative inhibitors of p53 and pRB not derived from viral elements. Thiscell line expresses (i) a truncated form of p53 (p53DD) that disruptsthe tetramerization of endogenous p53, (ii) a CDK4^(R24C) mutantresistant to inhibition by p16INK4A and p15INK4B (the major negativeregulators of CDK4) and (iii) cyclin D1. The effects of theseRAS^(V12)-selective compounds at a range of concentrations were testedin these cells, BJ-TERT/p53^(DD)/CDK4^(R24C)/D1/ST/RAS^(V12) (namedBJ-DRD) cells. These compounds were found to be active in this cell line(Table 2). Thus, these compounds (including erastin) are effective intumor cells transformed without viral proteins (other than ST). Inaddition, these compounds were tested in a second clone of BJ-derivedengineered tumor cells (FIG. 2) and found that they were effective inthis cell line as well.

BJ-TERT/LT/ST/RAS^(V12) cells grow more rapidly than BJ-TERT/LT/ST cellslacking RAS^(V12). Thus the activity of each compound was measured inBJ-TERT/LT/RAS^(V12)/ST cells, which were engineered independently andcontain cDNA constructs for LT and ST (whereas BJ-TERT/LT/ST/RAS^(V12)cells contain the genomic LT construct), and in BJ-DRD cells (describedabove). Truly oncogenic-RAS-selective lethal compounds should be equallyactive in all 3 of these cell lines. BJ-TERT/LT/ST/RAS^(V12) cells growmore rapidly than BJ-TERT/LT/RAS^(V12)/ST or BJ-DRD cells (FIG. 3).Thus, compounds that are targeting a mechanism dependent on the rate ofcell division should be more active in BJ-TERT/LT/ST/RAS^(V12) cellscompared to BJ-TERT/LT/RAS V12/ST or BJ-DRD cells. On the other hand,compounds that are acting in a cell-division-rate-independent mannershould be equally active in all three of these cell lines, as they allcontain HRAS^(V12).

Erastin was equally effective in the slower growing engineered cells,suggesting they act in a manner that is independent of the rate ofproliferation (e.g. FIG. 2). In addition, longer treatments and higherconcentrations had little effect on the viability of engineered cellslacking RAS^(V12), confirming the qualitative nature of theirselectivity (see Dolma et al⁸).

Voltage Dependent Anion Channels:

In certain aspects, the invention provides that one of theseRAS-selective lethal compounds, which was named erastin, acts throughthe voltage dependent anion channels (VDACs). VDACs, also known asmitochondrial porins, are small membrane-spanning channels thatfacilitate the transport of ions and metabolites across membranes, mostnotably the outer mitochondrial membrane⁶¹. There are three human VDACgenes, VDAC1, VDAC2 and VDAC3, of which VDAC1 is the moststudied^(62, 63). The three gene products are ˜70% identical, and likelyhave distinct cellular and organismal functions; for example, Vdac1-nullmice are viable but have altered respiration in striated muscle⁶⁴,whereas Vdac3-null male mice are infertile, but otherwise healthy⁶⁵.Murine embryonic stem cell lines have been generated lacking each Vdacgene, demonstrating that individual Vdac genes are not essential forcell viability⁶³. In addition, mice have been generated lacking bothVdac1 and Vdac3, demonstrating that an organism can survive with justone of the three Vdac isoforms⁶³.

Although no atomic-resolution structure of a VDAC protein is available,these proteins have been proposed to adopt a beta barrel fold analogousto the bacterial porins, based on amino acid sequence similarity and CDspectra^(66, 67). Unlike bacterial porins, however, the eukaryotic VDACsare gated by membrane voltage in vitro. In the closed state, ions, butnot small molecules, can penetrate through VDAC pores. In the openstate, both ions and metabolites can pass through VDAC pores. Themechanism of channel gating in vivo is not established, although proteinregulators of VDAC activity are reported to exist⁶⁸. The amino-terminalsegment of VDACs has been proposed to negatively regulate channelconductance⁶⁹. In support of this hypothesis are the findings that (i)mutations in this region change voltage dependence in vitro⁷⁰, and (ii)truncation of part of this region causes loss of voltage dependence⁶⁹.Immunostaining suggests that the amino-terminal helix of VDACs pointstowards the intermembrane space^(71, 72). While VDAC1 has been found toexist in a large protein complex with a molecular weight of 2 MDa, VDAC2has been found to exist as a monomer, such as an oligomer, and possiblyin a small multi-protein complex with a molecular weight of 230 kDa⁷³.Human VDAC1 has been reported to be localized to the plasma membrane, inaddition to its primary localization in the mitochondrial outermembrane⁷⁴⁻⁸¹. VDAC1's role in the plasma membrane is enigmatic; it hasbeen proposed to function as an NADH:ferricyanide-reductase. VDACsinteract with hexokinase⁸², the permeability transition pore⁸³,inter-mitochondrial membrane contact sites⁸⁴, the mitochondrial proteinimport complex⁸⁵ and microtubule associated protein-2 (MAP-2)⁸⁶. VDACshave been reported to interact with BCL proteins and to participate inthe formation of the mitochondrial permeability transition pore thatfacilitates release of cytochrome c from mitochondria⁸⁷⁻⁸⁹. VDACs mayregulate access of metabolites to the mitochondrial inter-membranespace. In yeast, NADH is transported into mitochondria through yeastVDAC⁹⁰. Finally, VDAC permeability has been linked to cell survival⁹¹,demonstrating that regulated opening of VDACs occurs in a physiologicalcontext.

Cell Death Pathways:

There are at least three types of mammalian cell death: (i) apoptoticdeath, (ii) autophagic death and (iii) necrotic death^(92, 93).Apoptosis is an intrinsic death program⁹⁴ involving activation ofcysteine proteases (caspases)^(93, 94). Autophagic death involvesself-digestion of cellular material through formation of lysosome-likeautophagosomes⁹⁵. Necrosis is a passive death process that involves lossof cellular homeostasis⁹⁶. Markers of apoptosis (Table 1) includecleavage of PARP1 from 113 kD to 85 kD⁹⁷, staining of by Annexin V98,release of cytochrome c from mitochondria⁹⁹, cleavage of chromosomalDNA¹⁰⁰, TUNEL staining¹⁰⁰, margination of chromatin¹⁰¹, activation ofcaspases¹⁰² and decreased cell size⁹⁹ (Table 1, column 2). Markers ofnecrotic cell death include increased PARP activity⁹⁷, clumping ofchromatin^(92, 93, 96, 103), decreased intracellular[ATP]^(92, 93, 96, 103), increased cell size and formation of reactiveoxygen species⁹⁶ (Table 1, column 3). Markers of autophagic deathinclude autophagosomes^(95, 104, 105), sensitivity to 3-methyladenine(3-MA) and increased lysosomal activity (Table 1, column4)^(95, 104, 105). Overlapping phenotypes exist: it is possible toactivate apoptotic-like mechanisms without caspases, or mixedapo-necrototic death¹⁰³. Other possible death programs includeparaptosis and mitotic catastrophe¹⁰⁶⁻¹¹³.

TABLE 1 Markers of different cell death phenotype, including markers forthe oxidative cell death provided by the invention APOPTOTIC NECROTICAUTOPHAGIC OXIDATIVE MARKER DEATH DEATH DEATH CELL DEATH Poly(ADP)ribosecleavage to Increased unknown no cleavage polymerase 85 kDa formactivity no activation Annexin V increased no change No change nostaining DNA laddering yes no No no TUNEL staining yes sometimes No NoNuclear margination/ clumping, partial no changes morphologyfragmentation karyolysis condensation ATP no Large No change smalldecrease change/small decrease decrease Cell size decrease increaseincrease Caspase yes sometimes No No activation ROS generation sometimesyes No Yes Suppressed by yes no No No Z-VADfmk? Cytochrome c yessometimes No No release Mitochondrial — — — altered: morphologyenlargement, fusion of mitochondria Abbreviations: 3-MA:3-methyladenine; TUNEL: terminal deoxynucleotidyl transferase-mediateddUTP-biotin nick end labeling; ATP: adenosone triphosphate; ROS:reactive oxygen species; Z-VADfmk:N-benzyloxycarbnyl-Val-Ala-Asp(O-Me)-fluoromethyl ketone.

Erastin Activates Non-Apoptotic Cell Death:

Among oncogenic-RAS selective lethal (RSL) compounds, erastin isattractive as a drug. Erastin is synthetically accessible and has atleast 16-fold RAS-selective lethality. To define the mechanism of actionof erastin, a two-pronged strategy was taken which involves: (i) atop-down approach, which characterized the type of cell death caused byerastin, and (ii) a bottom-up approach, which identified direct bindingproteins for the erastin scaffold.

To characterize the type of cell death induced by erastin, the effect oferastin was tested alongside camptothecin and staurosporine, whichinduce apoptotic cell death³⁹. Apoptosis is characterized by alterationsin nuclear morphology, including pyknosis, karyorhexis and marginationof chromatin⁹³. Nuclear morphology of camptothecin-treated anderastin-treated BJ-TERT/LT/ST/RAS^(V12) cells was monitored usingfluorescence microscopy. Although karyorhexis and margination ofchromatin were visible in camptothecin-treated cells, no suchmorphological alternations were visible in erastin-treated cells (Dolmaet al⁸). Further supporting the notion that erastin-induced death isnon-apopotic were the observations that: (i) erastin does not induce DNAfragmentation (i.e. formation of a DNA ladder), (ii) that a pan-caspaseinhibitor (50 μM Boc-Asp(Ome)-fluoromethyl ketone¹³⁹) does not blockcell death induced by erastin, (iii) that erastin does not causeincreased Annexin V staining (see Dolma et al.), (iv) that erastin doesnot cause the appearance of a caspase 3 active fragment⁸, (v) that PARP1is not cleaved upon treatment of cells with erastin (FIG. 5), and (vi)that cytochrome c is not released from mitochondria upon treatment witherastin (FIG. 6). Apoptosis-inducing compounds (staurosporine and/orcamptothecin) were tested alongside erastin in all of these assays toconfirm the functionality of the assay. Unlike many anti-tumoragents¹⁴⁰, erastin does not activate apoptosis. Therefore, in certainaspects the invention provides a genotype-selective anti-tumor agentwhich induces cell death via a non-apoptotic mechanism of oxidative celldeath.

Erastin Induces Genuine Cell Death:

Viability was quantified using calcein AM and Alamar Blue.BJ-TERT/LT/ST/RAS^(V12) cells treated with erastin rounded up anddetached, failed to exclude the vital dye Trypan Blue, displayed a lossof mitochondrial membrane potential as assayed by the potentiometric dyeJC-1, and had a small cell size characteristic of dead cells. Loss ofviability induced by erastin was irreversible once completed, in thatBJ-TERT/LT/ST/RAS^(V12) cells treated with erastin for 24 hours wereunable to recover when re-plated in erastin-free medium. Thus, erastininduces rapid, irreversible, non-apoptotic cell death in aRAS^(V12)-dependent fashion.

Erastin Activates Oncogenic-RAS-Dependent Oxidative Cell Death:

In certain aspects, the invention provides that oncogenic cells, such asfor example, oncogenic cells caused by RAS^(V12) signaling, treated witherastin undergo a rapid, oxidative cell death process. To define thetype of cell death initiated by erastin, a suppressor screen wasperformed using a library of ˜2,000 biologically active compounds¹¹⁴(from the Annotated Compound Library). It was found that antioxidants(e.g. alpha-tocopherol, butylated hydroxytoluene and beta-carotene)prevented erastin-induced death (FIG. 7).

Moreover, an oxidizing species were detected in response to erastintreatment in BJ-TERT/LT/ST/RAS^(V12) cells, but not in BJ-TERT cellslacking RAS^(V12) (FIG. 8; in certain experiments, BJ-TERT was used as acontrol cell line because the Small T oncoprotein does cause modestsensitivity to erastin, possibly by activating the RAS-MAPK pathway).The oxidizing species do not cause PARP cleavage, cytochrome c releaseor other hallmarks of apoptosis (FIGS. 5, 6). Thus, erastin-induced celldeath appears to involve a direct oxidative death. The mechanism oferastin induced cell death is in contrast to other anti-tumor agentsthat induce the formation of oxidative species along with activation ofapoptotic death.

Several observations suggested that erastin-induced oxidative speciesoriginate in mitochondria. First, antimycin, a mitochondrial complex IIIinhibitor^(141, 142) and 2-methoxyestradiol (2-ME), a superoxidedismutase inhibitor^(143, 144) both partially suppressed erastin-inducedcell death (Table 3, note that BJELR is a shorthand forBJ-TERT/LT/ST/RAS^(V12) cells). Both compounds act upstream ofmitochondria-generated hydrogen peroxide and hydroxyl radical (potentialoxidative species). However, melatonin, a peroxynitrite scavenger¹⁴⁵,did not affect erastin-induced cell death, suggesting peroxynitrite isnot involved.

Peroxisome proliferators (ciprofibrate, ciglitazone and clofibrate) andxanthine oxidase inhibitors (oxypurinol and allopurinol) did not affecterastin-induced cell death. Lipoxygenase inhibitors, prostaglandins,arachidonate esters and acids, and thromboxane receptor antagonists hadno effect on erastin-induced cell death, suggesting lipoxygenases andarachidonic acid pathways are not involved. In addition, verapamil (anMDR pump inhibitor) had no effect on erastin sensitivity, suggesting MDRactivity is not involved in the differential sensitivity of cells toerastin.

Erastin Inhibits Growth of Tumor Cell Lines with Activating Mutations inNRAS, KRAS or BRAF:

Given that oncogenic RAS activates at least four downstream pathways,analysis was performed to identify which downstream effects werenecessary for erastin sensitivity. In certain embodiments, the inventionprovides that erastin inhibits growth and kills genuine tumor cells withRAS mutations. In other embodiments, the invention provides measures ofthe dose-response of 29 tumor cell lines to erastin, wherein there is atleast 50% inhibition of viability in 18 of the cell lines (Table 4).Numerous sarcoma-derived tumor cell lines were sensitive to erastin,consistent with the fact that erastin was discovered in an engineeredtumor cell line created from human fibroblasts.

In non-limiting examples: HT1080 fibrosarcoma cells, with a knownactivating mutation in NRAS¹⁴⁶, were sensitive to erastin; Calu-1 lungcarcinoma cells and MIA PaCa-2 pancreatic cancer cells, with knownmutations in KRAS, were sensitive to erastin; A673 cells, with a knownV599E activating mutation in BRAF¹⁴⁷, were sensitive to erastin (FIG.11). BRAF is a direct target of RAS proteins. For some cell lines, it isnot known whether they have activating mutations in RAS pathwayproteins, or whether they harbor other mutations that activate theRAS-RAF-MEK-MAPK pathway. Activation of RAS has even been observed inthe absence of direct RAS mutations¹⁴⁸. Thus, for some cell lines, itmay not possible to directly correlate erastin sensitivity with RASmutation status.

For cell lines with known activating mutations in RAS pathwaycomponents, the necessity of such mutations for erastin sensitivity canbe tested. To determine whether the activating mutation in BRAF in A673cells influences erastin sensitivity, short-hairpin-RNA-expressingplasmids targeting either BRAF mRNA or, as a control, luciferase (LUC)mRNA were created. Stably-transfected cell lines containing theseconstructs were generated and cell line sensitivity to erastin wasmeasured (FIG. 11). A673 cells containing the control LUC-shRNAconstruct were sensitive to erastin, but A673 cells containing aBRAF-targeted shRNA were resistant to erastin. This effect was confirmedusing a second shRNA construct targeting BRAF, and again there wasresistance to erastin when BRAF was knocked down.

In addition, a cDNA construct expressing BRAF that is not targeted bythe shRNA was used to demonstrate the specificity of the shRNAconstructs. In this set of experiments, A673 cells were co-infected witha BRAF^(V599E) expression vector and a BRAF-shRNA expression vector,wherein the co-expression of the non-targetable BRAF mutant restoressensitivity to erastin. These experiments confirm that knockdown ofmutant BRAF causes resistance to erastin. As a control, all theshRNA-containing cell lines were equally sensitive to the cytotoxiccompounds doxorubicin and phenylarsine oxide, demonstrating no change inoverall drug sensitivity.

In further support of the notion that the RAS-RAF-MEK pathway sensitizestumor cells to erastin, three different MEK1/2 inhibitors were found tosuppress erastin's lethality in two different cell lines (FIG. 12). Inanother aspect, there is correlation between phospho-ERK1/2 abundanceand erastin sensitivity in sarcoma cell lines (correlationcoefficient=0.41). Therefore, the RAS-RAF-MEK pathway is an importantfactor in determining sensitivity to erastin.

Identification of VDACs as Erastin-Binding Proteins:

To identify direct protein targets of erastin, erastin analogs weresynthesized that could be linked to a solid-phase resin. Replacement ofthe p-chloro substituent in erastin with an aminomethyl group resultedin an analog (erastin A3, FIG. 14) that retained the ability toselectively kill BJ-TERT/LT/ST/RAS^(V12) cells. Replacement of thep-fluoro group in erastin B1 (an analog of erastin with nearly equalactivity, selectivity and potency) with an aminomethyl group resulted inan analog (erastin B2, FIG. 14) lacking activity in cells.

Erastin A3 and erastin B2 were immobilized on resins to identifyproteins that interact with the A3 resin but not the B2 resin. UsingBJ-TERT/LT/ST/RAS^(V12) cell lysates, all three isoforms of the humanmitochondrial voltage-dependent anion channels (VDAC1, VDAC2 and VDAC3)were identified on the A3 resin, and some VDAC1 was identified on the B2resin. Using BJ-TERT cell lysates, a small amount of VDAC1 wasidentified on the A3 resin, but no VDAC was identified on the B2 resin.No VDAC proteins were identified on a control resin lacking any erastinanalog. It thus appears that erastin A3 interacts more productively thanerastin B2 with VDAC2 and VDAC3. Moreover, all three VDACs wereidentified with higher confidence from BJ-TERT/LT/ST/RAS^(V12) celllysate, suggesting VDACs are expressed at a higher level in these cells.Higher level of VDACs expression in BJ-TERT/LT/ST/RAS^(V12) wasconfirmed (see FIG. 10). The finding that erastin pulls downmitochondrial proteins (VDACs) was consistent with the previous findingshowing that erastin induces a mitochondria-driven oxidative death.

VDAC Proteins are Unregulated by Oncogenic RAS Signaling:

In certain aspects, the invention provides that altered expression ofVDACs contribute to erastin sensitivity. To determine whether VDACs areupregulated in response to oncogenic RAS signaling, VDAC abundance wasmeasured, using an antibody that recognizes all three isoforms, in theBJ cell series (primary BJ cells, BJ-TERT cells, BJ-TERT/LT/ST cells,and BJ-TERT/LT/ST/RAS^(V12) cells). In the presence of oncogenic RAS,total VDAC protein was increased about four-fold (FIG. 10). There was noincrease in mitochondria number in BJ-TERT/LT/ST/RAS^(V12) cells asmeasured by a flow cytometric assay, suggesting the greater abundance ofVDAC proteins is due to specific upregulation of these proteins and notdue to increased mitochondrial biogenesis. Thus, in certain aspects theinvention provides connection between oncogenic RAS proteins and VDACproteins, including VDAC protein expression. In certain embodiments, theprotein level of VDAC1, VDAC2, VDAC3, or any combination thereof isincreased. In other embodiments, the mRNA level of VDAC1, VDAC2, VDAC3,or any combination thereof is increased. In certain aspects, theinvention provides an increased level of VDAC proteins, or mRNA as abiomarker to identify a tumor cell whose growth or viability can beinhibited by an agent which induces oxidative death. Non-limitingexamples of such agents are erastin, and its active analogues. Incertain embodiments, the tumor cell is derived from a subject whosuffers from a tumor. In certain embodiments, the biomarker is anincreased protein level of VDAC1, VDAC2, and/or VDAC3, or any isoform,or any combination thereof, in a tumor cell compared to syngeneic orisogenic cell.

In certain embodiments, there is loss of mitochondrial membranepotential in >70% of BJ-TERT/LT/ST/RAS^(V12) cells after 13 hours oferastin A1 treatment (JC-1 stain), and morphological changes inmitochondria examined by EM, consistent with the notion that erastininduces mitochondrial dysfunction.

In certain aspects, the invention provides that erastin acts by again-of-function mechanism and that cells with more VDAC proteins aremore sensitive to erastin. A gain-of-function mechanism operates incells which have increased levels of topoisomerase I and are thus moresensitive to camptothecin¹⁴⁹. In certain embodiments, upregulation ofVDAC proteins, VDAC1, 2, and/or VDAC3 by nucleofection caused anincrease in sensitivity to erastin, wherein the measured increase isabout two to three-fold (FIG. 32 panels (e) and (f)). A controlnucleofection process did not change the potency (i.e. the IC₅₀) ofpodophyllotoxin, a microtubule depolymerizer that acts through anunrelated mechanism. In other embodiments, cell lines can be stablytransfected with vectors expressing VDAC1, 2, and/or 3. Thus, increasedVDAC expression leads to erastin sensitivity which is consistent with again of function mechanism. Furthermore, a knockdown of VDACs causeserastin resistance.

Knockdown of VDACs Causes Erastin Resistance:

A lentiviral short hairpin (shRNA) construct was used to reduceexpression of VDAC proteins through RNA interference. Vector (pLKO.1)was used to generate 90,000 shRNA constructs targeting more than 18,000human and mouse mRNAs¹²⁵. This vector has been validated as an effectivemeans of knocking down many mRNAs in human and mouse cells, withoutinducing an interferon response. All constructs generated are sequencedand several hundred have been verified for their ability to knock downtheir intended mRNA target¹²⁵.

Five lentiviral pLKO.1-based constructs targeting each VDAC isoform werecreated. These constructs were tested for their effect on erastinsensitivity and the results showed that VDAC2-targeted andVDAC3-targeted shRNAs caused significant resistance to erastin. Thesefindings were consistent with the pulldown experiment, in that they bothindicated a preferential role for VDAC2 and VDAC3 in mediating erastin'seffects. These constructs cause knockdown of their specific isoformtargets at the mRNA and protein level (FIG. 15A). In certainembodiments, reducing expression of VDAC3 causes complete resistance toerastin. In other embodiments, reducing expression of VDAC2 causespartial resistance to erastin. This is consistent with again-of-function model and provides evidence that VDACs are functionallyimplicated in the erastin-induced cell death mechanism.

Iron Chelators Suppress Erastin Lethality:

Given that erastin-induced death is oxidative, it is likely that Fe²⁺ isnecessary for erastin's lethality. Fe²⁺ reacts with peroxides in acatalytic cycle through Fenton chemistry to generate hydroxyl radicalsthat react with proteins, lipids and nucleic acids. The effect of ironchelators, such as for example, the iron chelators shown in FIG. 30, onerastin's lethality were evaluated in BJ-TERT/LT/ST/RAS^(V12) cells.Iron chelators completely suppressed erastin's lethality (FIG. 13).These data show that Fenton chemistry is involved in erastin-inducedoxidative death, and that iron is necessary for erastin's lethality.

In certain aspects, the invention provides that erastin interacts with aVDAC-containing mitochondrial complex to induce mitochondrialdysfunction, release of oxidative species and cell death vianon-apoptotic mechanism. This mechanism is selective for cells withactivated RAS or RAF signaling, because oncogenic RAS/RAF upregulateVDACs (FIG. 10). Thus, cells with greater RAS, RAF or MEK activity havean increased pool of VDACs and are more susceptible to compounds thatdysregulate VDAC function. In certain embodiments, the dysregulation offunction can be by locking VDACs in an open conformation and causingexcessive respiratory activity. Because RAS^(V12)-expressing cells aremore glycolytic, they also accumulate higher levels of NADH, which wouldnormally affect VDAC closure; it is likely that erastin prevents thiseffect, leading to excessive respiratory activity and oxidative species.

Preclinical Assessment of Erastin:

The stability of erastin in mouse and human liver microsomes and inmouse and human plasma was assessed. In both human and mouse livermicrosomes, after 1 hr, ˜30% of erastin was converted to its primarymetabolite (the O-de-ethylated product). In both human and mouse plasma,only ˜10% of erastin was lost after five hours. Thus, erastin hassufficient metabolic and plasma stability to be tested in vivo in mice.

Measuring the Effects of Erastin on VDAC1, 2 and 3 In Vitro:

Overexpression and purification of the three human VDAC isoforms. HumanVDAC1, VDAC2 and VDAC3 can be overexpressed and purified^(89, 150-152).cDNA clones for the human VDACs are cloned, using PCR, restrictiondigests and sequencing to verify each clone, into the E. coli expressionvector pET-15b (Novagen). Amino-terminally hexahistidine-tagged VDACfusions proteins are produced in BL21 cells and purified from inclusionbodies, as described for S. cerevisiae VDAC purification from E. coli¹⁵². The pET vectors contain a thrombin-cleavage site, allowing optionalremoval of the affinity tag after purification. Purity is assessed bySDS page, reactivity with both N- and C-terminally directed antibodies,MALDI-TOF MS, HPLC, CD and voltage-dependent gating in lipid bilayers.For example, with VDAC3, the cDNA clone was transformed into E. coli,expression was induced with 0.5 mM IPTG and cells were grown overnight.After pelleting the cells, the resuspended pellet was incubated withlysozyme and TX-100. The resulting lysate was sonicated and centrifuged,washed and resuspended in solubilization buffer (100 mM NaCl, pH 8.0Tris-HCl, 6M Gdn-HCl). After removal of cell debris by centrifugation,the supernatant was applied to Ni affinity columns (BD biosciences),drained by gravity flow, and eluted with solubilization buffercontaining 50 mM imidazole. After adding LDAO (Sigma) to a finalconcentration of 2%, Gdn-HCl was dialyzed against storage buffer. Theresulting protein was pure on a Coomasie-Blue-stained SDS gel. VDAC1 andVDAC2 are purified in an analogous manner. All three VDAC proteins canbe fully characterized biochemically after the purification.

In a non-limiting method for native VDAC purification, recombinant VDACproteins can be isolated, and purified, from S. cerevisiae harboringeach murine or human VDAC isoform in place of the yeast VDAC, using aprotocol known in the art¹⁵³. In a non-limiting embodiment, murine andhuman VDAC2 and 3 were purified from yeast strains. Isolation ofmitochondria was as described by Daum et al. (Lipids of mitochondria.Biochim Biophys Acta. 822(1):142 (1985)), except cells were lysed usinga Dounce homogenizer after Zymolyase treatment. The mitochondrial pelletwas then lysed by incubating in a 50 mM Tris pH 7.5, 2.5% TX-100solution for 30 minutes with gentle shaking and then by centrifuging at27000 g-s to remove debris. The supernatant was run on a Sepharose Q FFcolumn (Amersham) using a NaCl (0.1-1M) gradient. The appropriatefractions were collected and concentrated using Centricon 10 (FisherScientific) tubes. The purity was analyzed by SDS-PAGE, reactivity withanti-VDAC antibody and mass spectrometry. Murine VDAC1 can be purifiedin a similar manner. This procedure typically yields up to 1 mg ofnative VDAC proteins that have not been refolded, which is advantageousfor lipid bilayer experiments.

In other embodiments for VDAC protein purification, mitochondrial outermembranes can be isolated from human cells lacking some andoverexpressing specific VDAC isoforms. In one embodiment, stablytransfected cell lines overexpressing VDAC1 (using zeocin and pcDNA3)can be generated, wherein in certain embodiments these cell lines cancontain short hairpin RNA (shRNA) constructs that eliminate expressionof VDAC2 and VDAC3. Selection of these cell lines can be accomplishedusing puromycin. In another embodiment, stably transfected cell linesoverexpressing VDAC2 (using zeocin and pcDNA3) can be generated, whereinin certain embodiments these cell lines can contain short hairpin RNA(shRNA) constructs that eliminate expression of VDAC1 and VDAC3.Selection of these cell lines can also be accomplished using puromycin.In another embodiment, stably transfected cell lines overexpressingVDAC3 (using zeocin and pcDNA3) can be generated, wherein in certainembodiments these cell lines can contain short hairpin RNA (shRNA)constructs that eliminate expression of VDAC2 and VDAC1. Selection ofthese cell lines can also be accomplished using puromycin.

cDNA clones for human VDAC1, VDAC2 and VDAC3 and five shRNA clonesspecifically targeting each VDAC isoform, i.e. 15 shRNA constructs totalin the pLKO.1 lentiviral shRNA vector, have been constructed, some ofwhich completely eliminate expression of each isoform (See, e.g., Table7). qPCR and 2D gels can confirm that the HT1080 fibrosarcoma-derivedclones transfected with shRNA vectors, indeed express a single VDACisoform. Outer mitochondrial membranes from such cell lines can beisolated using established methods^(74, 153).

Measurement of Erastin Isoform-Binding Specificity Using SPR andCalorimetry:

In certain embodiments, surface plasmon resonance (SPR) can be used tomeasure the affinity of the interaction between an agent which inducesoxidative cell death, such as for example erastin, and any of theisoforms of the VDAC proteins. Purified, hexahis-tagged, recombinantVDAC protein can be immobilize on a Biacore sensor chip using antibodycapture, with an anti-his antibody, and the change in SPR signal in thepresence of erastin can be measured. Native, non-refolded VDAC proteins,which, of example, can be derived from yeast strains harboring murine orhuman VDAC isoforms knocked into the yeast VDAC locus, can beimmobilized on a sensor chip using an Abcam pan-anti-VDAC antibody.Proteins such as GST, avidin and bovine serum albumin can serve asnegative protein controls. Erastin B1 and camptothecin, or any otherunrelated compound can be used as negative small molecule controls. Touse SPR, VDAC proteins can be solubilized in a buffer containing 20 mMTris (pH 7.0), 0.1 M (NH₄)₂SO₄, 10% glycerol, protease inhibitor tabletand 1% lipid/detergent mixture drawn from various combinations oflipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine),DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) and DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine). The solubilized protein canbe captured on an anti-his antibody immobilized on a CM4 Biacore sensorchip, or an anti-VDAC antibody immobilized on a sensor chip. A Biacore3000 optical sensor can be used to perform thesemeasurements^(154, 155). Erastin A3 can be immobilized on a Biacore chipand the binding of solubilized VDAC protein to the chip can be measured.This would provide a larger signal because of the greater molecularweight of VDAC relative to erastin. SPR can be preferable for initialstudies because it uses less protein than calorimetry, SPR can useprotein in the range of 100 μg versus mg quantities for calorimetry.

In another embodiment, isothermal titration calorimetry (ITC) can beused to measure the molar enthalpy (ΔH) and equilibrium binding constant(K_(B)) for the interaction between an agent which induces oxidativecell death, such as for example erastin, and any of the isoforms of theVDAC proteins. Calorimetry is the gold-standard method for measuringbinding because it does not suffer from artifacts of surface chemistrythat can complicate SPR binding data. From the calorimetry data, theGibbs free energy of binding (ΔG=−RT In K_(B)) and the entropy ofbinding (ΔS=(ΔH−ΔG)/T) can be calculated, where R is the ideal gasconstant and T is the temperature in the experiment^(156, 157). Byperforming this experiment at multiple temperatures, the change in heatcapacity upon binding (at constant pressure), ΔCp, can be calculated.Thus, these experiments allow characterization of the thermodynamicparameters associated with binding of erastin to each VDAC isoform. Ascontrols, the binding of an agent which induces oxidative cell death,such as for example erastin, to several other proteins can be tested,including glutathione-S-transferase (GST), avidin and bovine serumalbumin.

In other embodiments, mutations in VDACs that are predicted to disruptbinding, based on a homology model and docking experiments can bedesigned. VDAC mutant proteins of any one of the isoforms can beexpressed, purified, and tested for their binding to an agent whichinduces oxidative cell death, such as for example erastin. It isrecommended that the concentration of protein in an ITC ligand-bindingexperiment be at least 10 times the K_(B) estimate¹⁵⁶. Given thaterastin has a potency of 1 to 5 μM in several tumor cell lines, this isan upper limit on K_(B). It is desirable to have 20 μM VDAC protein insolution when erastin is added in the ITC experiment to ensure all addederastin is bound to target protein. Given that the sample cell requires1.4 mL, 28 nmol of each VDAC, or ˜1 mg of each VDAC protein are neededfor each calorimetry experiment. Thus, about 5 to 10 mg of each VDACisoform is necessary for this set of experiments. This is achievablegiven published VDAC expression protocols¹⁵². For these experiments,erastin can be used as the experimental compound and erastin B1 as anegative control that is not expected to bind to VDAC proteins with highaffinity, given its lack of lethality. Microcal Omega IsothermalTitration Calorimeter, which consists of an Omega reaction cell (T115),control module (T106) and nanovoltmeter (059), can be used for theseexperiments.

In another embodiment, there is provided a method for determiningwhether there is a change in the fluorescence of any of the VDACproteins upon incubation with an agent, such as for example erastin. Iferastin binds near a hydrophobic residue (Trp, Tyr or Phe), it ispossible that the binding would cause a change in the fluorescence ofVDAC that would allow us to determine the binding constant for thisinteraction. This would readily be detected using a fluorescencespectrometer. In another embodiment, a ³H-labeled erastin analog can becreated and used in a radioligand binding assay. The ³H-labeled erastinanalog can be created by acetylating erastin A3 with [H³]-CH₂COCl.Erastin A3 is the affinity analog used to purify VDAC, and ³H-labelederastin analog can be used in a charcoal precipitation or filter-bindingassay.

Measurement of Effect of Erastin on VDAC-Mediated Transport In Vitro:

In certain embodiments, the effect of agent, such as for exampleerastin, on VDAC-mediated transport in reconstituted liposomes and lipidbilayers can be determined. The flow of ATP through each VDAC isoformdetermined in the presence of erastin or erastin B1, can be used as anegative control. A method for measuring the flux of ATP through VDACchannels reconstituted into planar phospholipids membranes has beenreported¹⁵⁸. In a certain embodiment of this method, VDAC channels areopen at low voltage (<10 mV) and closed at higher voltages. The methodinvolves forming lipid bilayer membranes with a 1% solution ofdiphytanoylphosphatidyl choline and cholesterol in hexane using amodified Montal-Mueller technique⁵⁹. VDAC proteins in 1% Triton X-100are added to one side of the planar lipid bilayer; subsequently, aconcentrated ATP solution is added to the same (cis) side. Aliquots areremoved from the trans side and mixed with a D-luciferin/luciferasesolution; light output is determined using a luminometer; the [ATP] onthe trans side can be calculated as a function of time using acalibration curve. In certain embodiments, this method can be used todetermine whether an agent, such as for example erastin, accelerates orimpedes the rate of ATP flux through each VDAC isoform. In certainembodiments, the experiments can be performed as a function of membranevoltage to determine whether erastin increases ATP transport atvoltages>10 mV. In certain aspects, the invention provides that erastin,or any other agent that induces oxidative cell death, increases ATPtransport at voltages>10 mM. An agent which increases the rate of ATPtransport is an agent which induces oxidative cell death.

In other embodiments, the invention provides a method which can measurethe effect of an agent, such as for example erastin, on VDAC-mediatedtransport of NADH across the mitochondrial outer membrane. In anon-limiting example, a mitochondria containing fraction can be isolatedfrom yeast expressing a single, specific VDAC isoform, such as forexample human isoforms VDAC1, 2, or 3, or any homologues, or mutantversions thereof, in place of yeast VDAC. Using this mitochondriacontaining fraction, the rate of NADH oxidation can be measured. In thissystem, when NADH is added to isolated mitochondria, or a mitochondriacontaining fraction, NADH is transported across the mitochondrial outermembrane in a VDAC-dependent manner¹⁵³. The rate of transport can bedetermined by measuring the rate of NADH oxidation by theinner-mitochondrial-membrane-protein NADH dehydrogenase.

Mitochondria with a disrupted outer membrane transport NADH faster thanyeast mitochondria containing murine or human VDAC1, VDAC2 or VDAC3,suggesting that VDAC is rate-limiting for NADH transport across theouter mitochondrial membrane. In fact, the rate of transport of NADHthrough the murine VDAC isoforms is VDAC1>VDAC2>VDAC3. In certainembodiments, this method can measure the effect of an agent, such as forexample erastin, on the rate of NADH transport across the outermitochondrial membrane in mitochondria, expressing human VDAC isoforms,isolated from yeast. In other embodiments, this method can be used toidentify an agent which induces oxidative death. An agent whichincreases the rate of NADH transport, is an agent which inducesoxidative cell death. A non-limiting example of such an agent iserastin.

In other embodiments, cellular fractions containing mitochondria can beisolated from human cells, including but not limited to any of the tumorcell lines as described herein. In certain aspects the inventionprovides, that erastin causes increased flux through the outermitochondrial membrane when VDAC3 is present. In certain embodiments,the invention provides that erastin causes increased flux through theouter mitochondrial membrane when human VDAC1, 2 or 3 is expressed inyeast. In other embodiments, expression levels of VDAC1, 2 or 3 inyeast, can be modulated to determine the effect of VDAC protein levelson the rate of NADH transport, in the presence or absence of erastin.

In other embodiments, a method can measure the effect of an agent, suchas for example erastin, on the rate of NADH transport across the outermitochondrial membrane in mitochondria isolated from any suitable tumorcell line, such as for example HT1080 cells, which express only VDAC1,VDAC2 or VDAC3. These cell lines can be created by stably transfecting,e.g., HT1080 cells with a cDNA vector containing a specific VDAC isoformand shRNA vectors targeting the other VDAC isoforms. cDNA expressionconstructs for human VDACs can be created. shRNA constructs can becreated in a lentiviral backbone¹⁶⁰ (pLKO.1) targeting VDAC1 (fiveconstructs), VDAC2 (five constructs) and VDAC3 (five constructs). In anon-limiting example, the ability to down regulate VDAC expression of atleast one construct for each isoform was demonstrated. Theisoform-specific pattern of expression can be determined using qPCR and2-D gels. Once cell lines which express only specific VDAC isoforms areobtained, mitochondria containing fractions can be isolated, and therate of NADH oxidation in intact mitochondria versus mitochondria with adisrupted outer membrane¹⁵³ can be measured. In certain embodiments,measurements of the rate of NADH oxidation can be done to determine theeffect of an agent, such as for example erastin, on this rate of NADHuptake. In another embodiment, this method determines which VDAC isoformparticipates in NADH transport.

In other embodiments, the invention provides a method to measure theeffect of erastin on sucrose uptake by each human VDAC isoform inreconstituted liposomes^(89, 150). In this method, VDAC protein isreconstituted in liposomes using a sonic freeze-thaw method. Sucroseimport is determined by measuring liposomal swelling in the presence of50 mM sucrose; swelling is in turn measured by the amount of lightscatter at 520 nm. Sucrose import can also be determined by measuring[¹⁴C]-sucrose uptake^(89, 150). The above described functional assays,are non-limiting examples of assays that can be used to determinewhether an agent, such as for example erastin, effects functionalproperties of VDAC channels and whether there is isoform-specificity toany such effect.

Photocrosslinking of Erastin to Vdac Isoforms:

In certain aspects, the invention provides methods to determine how anagent, such as for example erastin, alters the functions of VDACproteins. In certain embodiments, erastin A3 can be coupled to abenzophenone-containing moiety to allow for photocrosslinking of erastinto each VDAC isoform. In a non-limiting example for synthesis and use ofthese photolabeled compounds, each VDAC isoform can be incubated with anerastin-benzophenone photo-reactive probe and inactive control probe inparallel, placed in optical glass cells (Starna, cat#1-SOG-10-GL14-S),purged with argon gas for 5 min and irradiated at 350 nm for 5-15 min ina Rayonet Reactor. After irradiation, the protein-erastin complex can bedigested with several different proteases and the resulting samplesubmitted for ES/LC-MS-MS analysis to identify peptide residues modifiedby this photolabel. There are multiple routes through which it will bepossible to use MS analysis to determine the site at which erastin iscross-linked to each VDAC isoform.

Creating and Testing Erastin-Binding-Defective VDAC Mutants:

In certain aspects, the invention provides methods to generate VDACmutants, which are defective in erastin binding. VDAC homology modelingand erastin in silico docking results as demonstrated in FIG. 17 can beused to create VDAC mutants that are functional, but that fail to bindto erastin. In certain embodiments, mutations can be introduced in theamino-terminal helical region of any one of the VDAC isoforms, such asfor example VDAC3 and the hinge region between the helix and the barrelbecause these sites would be logical erastin binding sites, given thatthe amino terminus negatively regulates conductance and that erastinacts via a gain of function which may involve locking the channel in anopen conformation.

In certain embodiments, mutations can be created by overlap extensionPCR, cloning of the cDNA into pcDNA3(zeo) and sequencing of the clone.To test the effect of each mutation, a stable cell line derived fromHT1080 cells can be created by transfecting and selecting zeocin.Subsequently, a lentivirus can be used to knock down expression of anyone of the VDAC isoforms, including VDAC3, and selection accomplishedwith puromycin to retain only knockdown cells. Viral titer and volumecan be adjusted as needed to confer>90% knockdown of these VDACisoforms. Thus, cells can express any one of the mutant VDAC isoforms,including but not limited to VDAC3 mutants, from the pcDNA3(zeo) vector.This expression can be confirmed by qPCR and 2D gel. Erastin sensitivityand cell viability can be measured by an automated Trypan Blue exclusionassay on a Vi-Cell, for each such cell line, to identify VDAC mutantsthat cause erastin resistance. For mutants that cause erastinresistance, the cDNA can be cloned into a bacterial expression vector,and the mutant protein can be expressed and purified, as describedabove, for the wild-type VDAC proteins. Once each mutant is purified,erastin binding can be measured by SPR and calorimetry, as describedabove. An assay can measure the effect of erastin on these mutants usinglipid bilayers to confirm a lack of gating by erastin. Suchcharacterization of erastin binding to VDAC demonstrates that binding oferastin to VDACs is necessary for erastin's lethality.

Hypothesis Connecting Oncogenic-RAS Signaling and Erastin Sensitivity:

In certain aspects, the invention provides that oncogenic-RAS-expressingcells are more sensitive to erastin because of two effects, both causedby the RAS-RAF-MEK pathway. In one aspect, Ras-expressing cells are moresensitive to erastin because of increased VDAC abundance. In anotherembodiment, Ras-expressing cells are more sensitive to erastin becausethey may have a need for a greater fraction of closed VDAC channels. Theincreased VDAC abundance may be due to increased transcription and/ortranslation of VDAC or decreased VDAC turnover. The increased VDACclosure in RAS-expressing cells may be due to increased glycolyticactivity in RAS-expressing cells that leads to (i) increased NADHabundance and (ii) increased activity of the electron transport chain,driving down the local pH in the mitochondrial inter-membrane space.Increased NADH abundance would lead to VDAC closure, as NADH causes VDACclosure in vitro and in isolated mitochondria^(161, 162). Low pH wouldlikely lead to VDAC closure in vivo, given that VDAC closure occurs inlow pH environments in vitro⁶⁷. Increased electron transport chainactivity would therefore lead to greater VDAC closure in vivo¹⁶³. Theoverall effect of these two factors would be to increase the pool ofclosed VDAC channels in cells with activated RAS-RAF-MEK signaling.

Erastin-induced locking of VDACs in an open conformation in cells withincreased levels of VDAC, including but not limited tooncogenic-RAS-expressing cells, would lead to excess electrogenicactivity of the electron transport chain, leakage of electrons todioxygen with concomitant production of hydrogen peroxide, whichencounters pools of free iron that in turn leads to Fenton chemistry andcatalytic production of reactive hydroxyl radicals. Thus, VDAC proteinsserve to homeostatically regulate activity of the electron transportchain, and dysregulation of this function leads to oxidative death dueto excess production of oxidative species.

Measure VDAC mRNA and Protein Levels with and without RAS, RAF and MEKSignaling:

To determine whether the RAS-RAF-MEK pathway leads to an increase inVDAC proteins without changing VDAC mRNA levels, VDAC1, VDAC2 and VDAC3mRNA and protein levels can be measured using any suitable methodincluding but not limited to quantitative RT-PCR (qPCR) and 2D gels.Probe-primer pairs were developed to measure all 3 VDAC mRNAs relativeto an internal standard. The primers used in this method amplify allthree isoforms and the internal control equally and are equallysensitive to changes in the input mRNA concentration. In certainembodiments, this method confirmed isoform-specific mRNA knockdown inengineered BJ cells and HT1080 cells. In other aspects, the inventionprovides a method to measure all 3 VDAC isoforms using 2D gels.

To test the effect of oncogenic KRAS and NRAS on VDAC mRNA and proteinlevels, VDAC mRNAs can be measured using qPCR in any cell line ofinterest, such as for example Calu-1 cells, which have mutant KRAS, andHT1080 cells, which have mutant NRAS, and in isogenic cells in whichKRAS or NRAS, respectively, can be knocked down using a lentiviralshRNA. pLKO.1 lentiviral shRNA constructs targeting KRAS and NRAS werecreated. Two KRAS-targeted constructs effectively knock down KRAS (FIG.9). These constructs can be transfected along with a vector expressingthe coat protein VSVG (pMD.G) and a packaging vector (pCMVdR.89) into293T cells using Fugene. Supernatant at 48 hours is collected and addedto the target cell lines (i.e. Calu-1 or HT1080). This protocol was usedto knock down each VDAC isoform using the pLKO.1 vector in HT1080 cells,BRAF in A673 cells and KRAS in Calu-1 cells. The effect of knockdown oneach VDAC mRNA can be measured using qPCR, as well as KRAS and NRAS toconfirm knockdown. In addition, protein lysates can be made, and run on2D gels, detecting the three VDAC isoforms with a pan-VDAC antibody. Theamount of each isoform can be quantitated relative to actin on the samegel using a LICOR Odyssey infrared scanner. As an additional control, asilent KRAS or NRAS mutant that is resistant to each of the effectiveshRNAs can be generated, and used to determine whether this resistantcDNA can reverse the effect of the shRNA. This will be a confirmationthat the shRNA is acting through the intended target (i.e. KRAS orNRAS). Similar experiments can be performed for BRAF. Overall, theseexperiments will determine the effect of HRAS, KRAS and NRAS signalingon VDAC mRNA and protein levels.

To test the effect of BRAF signaling, VDAC mRNA and protein levels(using pPCR and 2D gels, as described above) can be measured in A673cells (with an activated (V599E) mutant BRAF), and in isogenic cells inwhich BRAF is knocked down using a viral shRNA construct. As additionalcontrols, the effects of at least five additional shRNA constructstargeting BRAF can be tested, wherein the effects of each of these shRNAconstructs can be tested also in the presence of a BRAF cDNA containinga silent mutation that renders it resistant to each shRNA, and an shRNAconstruct targeting luciferase. Five additional lentiviral shRNAconstructs targeting BRAF were obtained, and two of them demonstratedknock down of BRAF at the protein and mRNA level. These experiments candetermine the oncogenic BRAF signaling on any of the VDAC isoform mRNAor protein levels.

To test the effect of MEK1/2 signaling on VDAC mRNA levels, VDAC mRNAand protein levels can be measured by any suitable method, including butnot limited to qPCR and 2D gels, in four cell lines (FIG. 12, HT1080,A673, Calu-1 and BJ-TERT/LT/ST/RAS^(V12)) that have been treated witheach of three structurally different MEK1/2 inhibitors. Non-limitingexamples of MEK1/2 inhibitors are provided in FIG. 31. These experimentscan determine whether MEK signaling affects mRNA or protein levels ofany of the VDAC isoforms. An effect of MEK inhibitors on VDAC protein ormRNA levels, can be confirmed and the specific MEK isoform responsiblefor this effect can be determined by obtaining or creating shRNAconstructs targeting MEK1 and MEK2 specifically. MEK1 and MEK2 functioncan be knocked down by similar protocols and control experiments asdescribed for RAS and RAF knockdowns.

Knockdown experiments can be controlled to ensure that shRNA induceknockdown of the desired target (KRAS, NRAS, BRAF or MEK1/2) at the mRNAand protein levels, and that related isoforms are not affected (i.e.including HRAS, CRAF and ARAF). MEK inhibitors should block MEK1/2phosphorylation of ERK1/2 substrates, and this can be confirmed bywestern blot with a phospho-specific ERK1/2 antibody. These experimentscan determine the role of RAS, RAF and MEK signaling (and specificfamily members) in modulating mRNA and protein levels of each VDACisoform.

Methods to Measure the Ratio of Open and Closed VDAC in Cells with andwithout RAS, RAF and MEK Signaling:

In certain aspects, the invention provides a fluorescently labeled VDACreporter construct of any one of the VDAC isoforms. In certainembodiments, the reporter construct is labeled with YFP, CFP, RFP, anyof the (Fluorescent Protein) FP optimized variants, or any othersuitable fluorescent label. In certain embodiment, reporter constructsare fully functional and active in vivo and in vitro, as measured by anysuitable assay which determines VDAC protein function. Methods andprotocols for creating fluorescent reporter proteins are well known inthe art. The FP can be fused to a VDAC isoform at any position in theVDAC protein, so long that the fluorescent VDAC reporter remainsfunctional. The FP can be fused to the VDAC isoform with or without aprotein linker sequence. In certain embodiments, a VDAC reporterconstruct comprises at least one FP. In other embodiments, a VDACreporter comprises two fluorescent proteins. The two FP can be identicalor different. In certain embodiments, the FP is fused to the N-terminus.In other embodiments, the FP is fused to the C-terminus.

In certain embodiments, fluorescently labeled VDAC proteins can be usedto monitor expression and localization of VDAC isoforms. In otherembodiments, fluorescently labeled VDAC proteins can be used in assaysto determine the effect of agents, including but not limited to erastin,on the expression, and/or stability of VDAC isoforms. Treatment witherastin induces disappearance of VDAC2 and 3 isoforms, as determined byWestern and 2D-gel electrophoresis, indicating that an agent such aserastin, which induces oxidative cell death, leads to disappearance ofits protein target. In certain aspects, the invention provides a methodfor identifying agents which induce oxidative cell death. In certainembodiments, the method comprises, contacting cells or mitochondrialcell fractions from cell with an agent, determining cell viability anddetermining whether VDAC protein levels remain unchanged or becomereduced in response to treatment with the agent. In other embodiments,the method comprises, contacting cells or mitochondrial cell fractionsfrom cell, which express a fluorescently labeled VDAC isoform, with anagent, determining cell viability and determining whether VDAC proteinlevels remain unchanged or become reduced in response to treatment withthe agent Contacting can be done in the presence or absence of a secondagent, wherein the agent inhibits formation of oxidative species inmitochondria, or the agent is an iron chelator, or the agent is anantioxidant. In certain embodiments, determining whether VDAC protein isreduced can be done by measuring the fluorescent signal due to thefluorescently labeled VDAC isoform. In other embodiments, determiningwhether VDAC protein is reduced can be done by any suitable method knownin the art, including but not limited to Western blotting, or 2D-gelelectrophoresis. An agent which leads to loss of cell viability, anddecrease in VDAC protein level, and/or the fluorescent signal due to aVDAC reporter is indicative of an agent which induces oxidative celldeath.

In certain aspects, the invention provides a method to measure the ratioof open to closed VDAC proteins. In certain aspects, the inventionprovides a fluorescent reporter construct that exhibitsconformation-dependent fluorescence that can be used to determinewhether a VDAC channel is in an open or closed conformation. VDACreporter constructs can be created in which yellow fluorescent protein(YFP) and cyan fluorescent protein (CFP) are fused to two differentpositions of VDAC, such that they create conformation-dependentfluorescent sensors. CFP and YFP containing fluorescent proteins havebeen used to create conformation-dependent fluorescent sensors¹⁶⁴. WhenCFP and YFP come into close proximity, they engage in fluorescenceresonance energy transfer (FRET), which can be detected by a change inthe fluorescence spectrum of the fusion reporter protein.

Sites within VDAC can be chosen to incorporate CFP and YFP, wherein thesites are chosen based on current models for sites that exhibit aconformational change upon opening. For example, the amino terminalhelix of VDAC has been implicated genetically as a negative regulator ofchannel permeability. One model for VDAC function posits that this aminoterminal loop sterically occludes the face of the channel when it isclosed^(163, 165). A second model involves movement of several strandsof the beta barrel out and up to the surface of the lipidbilayer^(166, 167). CFP can be fused to the VDAC amino terminus and YFPto different positions at the entrance of the beta barrel or on the Cterminus, to create a construct in which CFP is brought into proximitywith YFP when the channel closes. Thus, by creating a number ofdifferent CFP and YFP bearing constructs, such as for example about10-20 constructs, based on these two classes of models, CFP-VDAC-YFPreporter construct(s) that display(s) a change in fluorescence uponopening and closing can be identified.

In certain embodiments, the CFP-VDAC-YFP reporter construct can beexamined for fluorescence changes when the CFP-VDAC-YFP reporter is inlipid bilayers. In other embodiments, the fluorescence changes can bemeasured when the voltage dependent opening and closing of the VDACchannel is determined. The lipid bilayer setup is similar to the set up,which measures the flux of ATP through VDAC channels using aluciferase/luciferin solution on the trans side of the bilayer. These invitro experiments can identify a construct that exhibitsvoltage-dependent opening and closing of the VDAC channel as measured bythe changes in fluorescence emission of the fluorescent reporter. Suchvoltage dependent constructs can be created for all three isoforms(VDAC1, 2 and 3), enabling determination of the ratio of open/closedchannel for each VDAC isoform. In certain embodiments, such CFP-VDAC-YFPreporter constructs can be used to identify agents which increase theprobability that the VDAC channel is in an open conformation, asmeasured by a decreased or absent FRET signal. A non-limiting example ofsuch an agent is erastin.

To test CFP-VDAC-YFP reporter constructs in cells, constructs can becloned in the pLKO.1 lentiviral vector we have used previously todeliver shRNAs. The construct can be co-transfected into 293T cellsalong with a vector expressing the coat protein VSVG (pMD.G) and apackaging vector (pCMVdR.89), as described above and virus-containingsupernatant harvested and transferred onto target cells. Specifically,these constructs can be tested for RAS-pathway-dependent closing usingthe series of cells, including various knockdown cell lines, describedabove. To determine the effect of RAS signaling, the constructs can betested in any suitable cell line, for example but not limited toBJ-TERT/LT/ST/RAS^(V12) cells and BJ-TERT/LT/ST cells, Calu-1 cells,which have mutant KRAS, and derivative cells with mutant KRAS knockeddown, and HT1080 cells, and a derivative line with mutant NRAS knockeddown. For RAF signaling, we will test the constructs in A673 cells (BRAFmutant) and a derivative cell line with BRAF knocked down. To determinethe effect of MEK signaling, the constructs can be tested in anysuitable cell line, including but not limited toBJ-TERT/LT/ST/RAS^(V12), Calu-1, HT1080 and/or A673 cells, each treatedwith one of three different MEK1/2 inhibitors. In each of these cases,the assay can determine whether there is a change in the fluorescence ofthe CFP-VDAC-YFP construct when RAS-RAF-MEK signaling is active orinhibited. One form of a suitable negative control can be amitochondrial outer membrane targeted CFP-YFP fusion protein that lacksmost of the VDAC sequence. A suitable form of a positive control can bea constitutively open VDAC mutant as described herein. Erastin can betested to determine whether it causes VDAC opening in cells, as measuredby this CFP-VDAC-YFP fluorescent reporter. Cellular fractions containingmitochondria and/or purified mitochondria from cells expressing any oneof the CFP-VDAC-YFP reporters, including reporters with a VDAC mutationscan also be used. In assays using purified mitochondria or cellularfractions with mitochondria, NADH can be used as a control for inducingVDAC closure. In certain aspects, the invention provides methods whichdetermine whether the RAS-RAF-MEK signaling causes VDAC closure.

In other embodiments, a fluorescent VDAC reporter can be used as apharmacodynamic marker for erastin in mice. Stably transfected HT1080and Calu-1 cells harboring this fluorescent VDAC reporter, can begrafted in mice, which are treated with erastin or related analogs.After erastin treatment, the tumor can be excised and the fluorescenceof the VDAC reporter measured. This can ascertain whether erastin isgetting into the tumor xenograft and inducing the desired opening ofVDAC in the mouse model.

Knock Down Candidate MAPK Proteins and Measure Effects on Vdac Levelsand Closure:

To delineate the specific RAS-RAF-MEK pathway that regulates VDAC levelsand closure, the effect of knocking down candidate MAPK proteinsdownstream of MEK can be tested. The canonical RAS-MAPK pathway involvesa RAS-RAF-MEK1/2-ERK1/2 cascade¹⁶⁸. However, there are additional ERKproteins (ERK3-8) and other MAPK proteins (p38 and JNK proteins) thatcould potentially be involved. In addition, there might be other kinasesacting upstream or downstream of this cascade. The activity of humankinases and kinase-related proteins (about 850 proteins with predictedfunction) can be knocked-down using 4,250 lentiviral shRNA constructs.shRNA constructs are created and available from the RNAi consortium¹²⁵.The set of shRNAs targeting human kinases or kinase-related proteins canbe organized and maintained as a subset of the entire shRNA collection,wherein stocks of lentivirus for each of these 4,250 constructs aremaintained. These virus stocks are aliquoted in multiple 384-wellplates. To test the effect of knocking down each kinase on erastinsensitivity, the virus can be added to HT1080 cells, and after 24 h,selected by puromycin selection (each shRNA construct contains apuromycin resistance gene). After 1, 3 and 5 days (i.e. in parallelexperiments), erastin can be added, after 24 h, cell death/viability canbe determined by adding Alamar Blue to a final volume of 10%. For asubset of shRNAs, for example those targeting ERK1-8 and all thoseconfirmed to cause erastin resistance, the desired target mRNAknock-down can be examined by qPCR and western blotting. All candidateshRNAs can be validated by creating at least 3 shRNAs capable ofknocking down the same target mRNA>80% and confirming they all causeresistance. A silent mutation can be created in the target mRNA thatprevents knockdown, and tested whether this mutation restores erastinsensitivity. In all candidate cell lines with shRNAs that cause erastinresistance, the effect of knocking down the kinase activity on: VDAC1, 2and 3 mRNA and protein levels, and the effect on VDAC closure, can bemeasured as described herein. In certain aspects, the invention providesmethods to define additional kinases on the RAS-MAPK pathway that leadto increased VDAC protein levels and increased erastin sensitivity.

Determining the Subcellular Localization of Erastin-Binding Activity:

Although almost all of the VDAC proteins are localized to mitochondria,there is evidence that a small amount of VDAC1 is localized to theplasma membrane^(74-76, 81). Thus, it can be demonstrated that erastinbinds to mitochondrial VDACs, as opposed to plasma membrane VDACs. Toaddress this issue, radiolabeled erastin can be synthesized. In oneembodiment, a ³H-labeled erastin analog can be synthesized byacetylating erastin A3 with [H³]-CH₂COCl; A3 is an affinity analog usedto purify VDAC. In another embodiment, ¹²⁵I-labeled erastin can besynthesized, in which the label replaces the p-chloro substituent inerastin. In another embodiment, a ³H-labeled erastin analog can besynthesized in which the chiral methyl position is replaced with anacetamidoethyl group, this site tolerates larger groups such as apropylphthalamide without losing activity. In each embodiment, acharcoal precipitation assay can be used to measure binding of theradiolabeled analog to subcellular fractions (nucleus, cytosol,mitochondria and plasma membrane). In another embodiment, the method cancomprise a step for determining whether the binding can be competed witherastin itself. These studies will determine whether erastin-likecompounds bind to a mitochondrial target (i.e. mitochondrial VDAC).

Creating Mitochondria-DNA-Deficient Cells and Testing the Effect onErastin Sensitivity:

In certain aspects, the invention provides that erastin interacts withmitochondrial VDACs to cause mitochondrial dysfunction; disrupting theelectron transport chain should cause resistance to erastin. AntimycinA, a mitochondrial complex III inhibitor, causes resistance to erastin(Table 3). Another method that can determine whether erastin inducesmitochondrial dysfunction is to generate ρ⁰ cells, cells lackingmitochondrial DNA and therefore a functional electron transport chain,and test the effect on erastin lethality.

ρ⁰ cell derivatives for three different cell lines (HT1080, Calu-1 andBJ-TERT/LT/ST/RAS^(V12)) can be generated by treatment with 1.5 μg/mLditercalinium, as described^(169, 170), for 2 months. Single-cell clonescan be isolated and their mitochondrial DNA-deficiency confirmed by (i)Southern blot with a mtDNA probe, (ii) PCR analysis using primersderived from positions 8196-8215 and 8726-8707 of human mitochondrialDNA, and [³⁵S]-labeling of mitochondrial translation products using[³⁵S]-methionine and emetine, which inhibits cytoplasmic translation. Inaddition, ρ⁰ cells should be unable to grow in the absence of uridine orpyruvate. ρ⁰ cells also lack cytochrome c oxidase activity, which can beconfirmed using an established assay¹⁷¹. Medium from cell cultures in96-well plates is aspirated, 0.01% saponin is added in water topermeabilize cells, reduced cytochrome c is added, and catalase and 4 mM3,3′diaminobenzidine (DAB) in 0.1 mM sodium phosphate buffer are added.DAB is oxidized by the oxidized cytochrome c produced by the assay,resulting in a polymer that is detectable at 450 nm. By performing theexperiment in the presence or absence of KCN, the specificity of theassay can be monitored. The sensitivity of ρ⁰ cells to erastin can betested using an automated Trypan Blue exclusion assay, on a BeckmanVi-Cell, using a dilution series of erastin in replicate. This assay candetermine whether mitochondrial respiratory activity is necessary forerastin's lethality.

Methods to Determine the Effect of Expressing Mitochondria-TargetedCatalase and Ferritin on Erastin Sensitivity:

If erastin causes the appearance of hydrogen peroxide and Fe⁺² inmitochondria, then expression of mitochondria-targeted catalase orferritin is likely to cause resistance to erastin. In certain aspects,the invention provides methods for expressing mitochondrial ferritin andmitochondrial catalase in HT1080, Calu-1 and BJ-TERT/LT/ST/RAS^(V12)cells. A mitochondrially-localized catalase construct is previouslydescribed¹⁷². This construct can be cloned into pLKO.1, the lentiviralexpression vector used successfully to deliver shRNAs and GFP to thesecells. As controls, wild-type human catalase, which has a peroxisomallocalization signal, a nucleus-targeted catalase construct, which wasdescribed previously¹⁷², or GFP can be expressed. In each case, we willtransfect each construct into 293T cells along with a vector expressingVSVG (pMD.G) and a packaging vector (pCMVdR.89), harvest supernatantafter 48 h and infect three cell lines that are sensitive to erastin(HT1080, Calu-1 and HeLa). We will measure cell lethality using TrypanBlue exclusion on a Vi-Cell.

To test the role of free iron in erastin's mechanism of action,mitochondrial ferritin¹⁷³ or cytosolic ferritin (both heavy and lightchains) can be expressed in HT1080, Calu-1 and HeLa and their effect onerastin's lethality measured. These studies can determine whether ironand hydrogen peroxide are necessary for erastin's lethality, and whetherthese species are in mitochondria.

Methods to Create and Test Constitutively Open VDACs for their Abilityto Phenocopy Erastin:

In certain aspects, the invention provides that erastin locks one ormore VDAC proteins into an open conformation, causing dysregulated fluxof ions and metabolites through the outer mitochondrial membrane. Incertain embodiments, the invention provides methods for creating mutantsof each VDAC which mutants are constitutively open, and phenocopyerastin lethality.

Certain VDAC mutants have altered gating properties in vitro. Thevoltage sensor in VDACs consists of multiple lysine residues thatrespond to a transmembrane voltage potential by instigating largeconformational changes in the channel, rendering it poorly conducting toanions such as ATP/ADP⁷⁰. Single mutation of each of five differentlysine residues to glutamate (K19E, K46E, K61E, K65E and K84E) increasedthe voltage required to close the channel. Thus, VDAC proteins withmutations, single mutations or combinations of multiple mutations wouldhave a greater fraction of open channels when expressed in cells.Expressing these mutants in tumor cells can test the notion that openingVDAC channels leads to the lethality induced by erastin. Singlemutations or combinations of multiple mutations can be created in allthree VDAC isoforms using overlap-extension PCR, all resulting cDNAs canbe sequenced and cloned into pLKO.1¹²⁵. K→E mutant VDAC proteins can beexpressed in three tumor cell lines (HT1080, Calu-1 and HeLa) bytransfecting a packaging cell line (293T) along with a vector expressingthe coat protein VSVG (pMD.G) and a packaging vector (pCMVdR.89), andtransferring the supernatant to target cells. Viability can be tested at24, 48 and 72 h after infection using Trypan Blue (on a BeckmanVi-Cell). For constructs that cause lethality, the type of cell deathcan be characterized to determine if it phenocopies erastin: thedetermination can include (i) whether anti-oxidants and iron chelatorscan prevent mutant-VDAC-induced cell death, (ii) whether there arereactive oxygen species (ROS) which can be measured directly using flowcytometry with the ROS-sensitive compound dihydrodichlorofluorescein and(iii) whether hallmarks of apoptosis are activated or not. In certainaspects, the invention provides that opening of VDAC channels leads toan oxidative, non-apoptotic mode of cell death, which is the hallmark oferastin lethality.

Methods to Perform Large-Scale shRNA Suppressor Screens to DiscoverOther Regulators of Erastin Sensitivity:

The above-described experiments are targeted to the specific hypothesiswe currently hold regarding erastin's mechanism of action. Anothermethod of illuminating erastin's mechanism of action is to perform alarge suppressor screen for shRNA constructs that prevent erastinlethality. Such a screen can reveal that knockdown of VDAC mRNAs causesresistance to erastin, identifying these critical proteins in erastin'smechanism of action. Such a screen can illuminate: (i) proteinsdownstream of the RAS-RAF-MEK cascade that lead to increased VDACabundance (and therefore increased erastin sensitivity), (ii) factorsthat regulate the pool of free iron that is needed for erastin'slethality, (iii) proteins involved in regulating abundance of thecritical substrates whose gating by VDAC is altered in the presence oferastin, and (iv) pathways involved in detoxifying the oxidative speciesgenerated by erastin treatment.

To perform this shRNA suppressor screen, a collection of 90,000 shRNAclones in the pLKO.1 vector that was generated as part of the RNAiConsortium can be used¹²⁵. There is a high-throughput protocol forproducing plasmid DNA and lentivirus for this collection, and thisprotocol was validated in a screen for anti-mitotic shRNAs (see Moffatet al). To perform this screen, a co-transfection (with Fugene) can becarried out in 293T cells in a multi-well format using each pLKO.1 shRNAvector with packaging and envelope vectors. Supernatant can be harvestedafter 48 h, aliquoted and frozen. For the screen itself, about 3,000HT1080 cells per well of a 384-well plate can be seeded. The screen useda calibration of the relationship between cell number and Alamar Bluefluorescence and determination that 3,000 cells gives us a signal in themiddle of the dynamic range of the assay. The next day, one lentiviralshRNA stock is added to each well of the plate using a Beckman Biomek FXwith integrated Cytomat hotel (and enclosed in a BL2 Baker Bioprotect IIHood). The plates are incubated for 48 h to allow time for knockdown tooccur and for residual protein to turn over. A lethal dose of erastin (5μM) is added to all wells of the plate (except untreated control wells).After 20 h, Alamar Blue is added. Alamar Blue reduction is measured byexcitation at 530 nm and emission at 590 nm on a Victor3 (PerkinElmer)fluorescence plate reader. shRNA clones that cause>50% rescue oferastin-induced cell death can be retested to confirm their activity.shRNA can include shRNAs targeting mRNAs coding for proteins involved inoxidative stress, mitochondrial function, iron metabolism, deathsignaling and RAS signaling.

Once active shRNAs that suppress erastin's lethality are identified, thetarget mRNAs for each shRNA can be determined. At least five shRNAstargeting the same mRNA in parallel can be tested. Discovering more thanone shRNA targeting the same mRNA increases confidence that the putativetarget mRNA is in fact the correct target mRNA. In addition, knockdownof the target mRNA can be determined by qPCR, and of the correspondingprotein using western blotting. For the most effective mRNA targets,another step can determine that the target mRNA is responsible for theerastin resistance by creating a cDNA that contains a silent mutationthat renders the cDNA resistant to each shRNA construct. HT1080 cellscan be co-infected with each shRNA and the corresponding non-degradablecDNA, to determine whether this restored erastin lethality. Ifdegradation of a specific mRNA truly causes resistance to erastin, thenexpressing such an shRNA-resistant cDNA for the target mRNA shouldrestore erastin sensitivity. This shRNA suppressor screen will yieldcandidate mRNA (and corresponding proteins) that cause resistance toerastin. These can yield information on the pathways leading to erastinsensitivity and erastin-induced oxidative death.

In certain embodiments, the sensitivity to erastin, can be tested incell lines that are resistant to taxol and vinblastine. The sensitivityof CCRF-CEM parental, and taxol and vinblastine resistant derivativecells to erastin was tested. In certain aspects, the invention providesthat erastin is equally effective in inducing cell death in all 3 lines,CCRF-CEM parental, and taxol and vinblastine resistant derivative cell,with IC₅₀=11 μM, 7 μM and 8 μM, respectively, wherein taxol (IC₅₀=1 nM,560 nM, 1379 nM) and vinblastine (IC₅₀=1 nM, 93 nM and 370 nM) are noteffective in inducing cell death. Thus, cross resistance to erastin doesnot develop in taxol or vinblastin resistant cell lines. Erastinanalogues can be tested to ensure their effectiveness in inducing celldeath in tumor cell line which are resistant to taxol or vinblastin, orother ant-tumor agents.

Determine Pharmacokinetics of Erastin:

Pharmacokinetic parameters (AUC, t_(1/2) and C_(max)) in plasma and inxenograft tumors can be measured, using standard procedures. Briefly,heparinized blood samples can be collected at the following time points:5, 10, 20, 30, 60 min and 2, 3, 4, 5, 6, 8, 16 and 24 hours, followingadministration of erastin, or erastin analogues. Different routes ofadministration, such as for example IP, IV and PO, can be compared todetermine differences in pharmacokinetic and pharmacodynamic parameters.HPLC-UV and LC-MS can be used to measure analog concentrations at eachtime, relative to an internal standard. AUC, t_(1/2) and C_(max) can bedetermined using WinNonLin v 4.1 (Pharsight). These studies candetermine the optimal route of administration, the likely dosingschedule that will be needed, and can provide an estimate of the desiredtreatment dose. This information can be used in designing murinexenograft efficacy experiments with compounds selected for in vivotesting.

In certain embodiments, erastin was formulated at 150 mg/kg in 10% DMSO,20% Tween 80, 70% saline and 0.5 mL of this formulation was injected viaIV twice per day for four days in 3 nude mice. No gross toxicity, judgedfor example by body weight and behavior, was observed. In addition, theeffects of single dose IP injections of erastin in 100% DMSO up to 450mg/kg was tested and no overt toxicity was observed, suggesting erastinis relatively benign and non-toxic. Thus, PK experiments can beinitiated using the DMSO/Tween 80/saline formulation.

In Vivo Efficacy Testing of Erastin, or Erastin Analogs Using HumanTumor Xenoqrafts in Nude Mice:

To test the effect of erastin, or erastin analogs on tumor size in mice,six-week old athymic nude female mice (from NCI's Frederick CancerCenter) can be used. These experiments can be performed as perpreviously published work^(174, 175). HT1080, Calu-1 and A673 cells canbe tested for suitability in generating xenografts. When tumors reach 5mm in diameter, the mice can be distributed into treatment groupsrandomly. A stock solution of erastin or an analog thereof to be testedcan be prepared in DMSO/Tween 80 saline. The dose will depend on theresults of preclinical assessment, and can be in the range of 1-100mg/kg. Mice can be dosed up to 3× per day, according to the pre-clinicalassessment of the rate of drug elimination. After the dosing regimen hasbeen completed, for example but not limited to twice per day for fivedays, possibly repeated one or more times, the mice will be sacrificedusing Nembutal and CO₂ euthanasia. Tumor diameter can be determined bycaliper. Tumor size, expressed as tumor volume in cubic millimeters, canbe measured in untreated and several groups, treated with differentdoses of each analog and with a control such as paclitaxel ordoxorubicin.

In another embodiment, the invention is an erastin analog, such as acompound having formula I:

whereinR₁ is a hydrophobic or hydrophilic substituent, which is attached to oneor more positions of at least one carbon atom of the ring;R₂ is selected from is selected from H, C₁₋₈alkyl, C₁₋₈alkoxy, 3- to8-membered carbocyclic or heterocyclic, aryl, heteroaryl, C₁₋₄aralkyl,residues of glycolic acid, ethylene glycol/propylene glycol copolymers,carboxylate, ester, amide, carbohydrate, amino acid, alditol,OC(R₆)₂COOH, SC(R₆)₂COOH, NHCHR₆COOH, COR₇, CO₂R₇, sulfate, sulfonamide,sulfoxide, sulfonate, sulfone, thioalkyl, thioester, propylphthalimide,and thioether;R₃ is a C₂₋₈ alkoxy;R₆ is selected from H, C₁₋₈alkyl, carbocycle, aryl, heteroaryl,heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle, whereineach alkyl, carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, and alkylheterocycle may be optionally substituted withat least one substituent;R₇ is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl, aryl,carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, alkylheterocycle, and heteroaromatic may be optionallysubstituted with at least one substituent; oran enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.

All possible enantiomers, optical isomers, and diastomers of eachformula and compound recited herein are part of the invention, whetherthey are explicitly shown or not. In the present invention, the isomericforms of the compounds may be synthesized de novo. Alternatively, thespecific desired isomeric form may be separated from, e.g., a racemicsolution using conventional techniques, such as for example, gaschromatography. Moreover, the present application includes everypossible combination of each R group, whether explicitly identified ornot.

In one aspect of this embodiment, the hydrophilic substituent isselected from alcohols, amines, nitro, carboxylic acids, carboxylates,hydroxy, amides, sulfamides, sulfonic acids, sulfonates, sulfates,esters, thiol esters, ethers, thiols, thiolates, thiol ethers,morpholino, fluoroaromatics, piperazines, piperadines, phosphonates, andsalts thereof, and combinations thereof. Preferably, the hydrophilicsubstituent is selected from NH₂, NO₂, NCOCH₃, and combinations thereof.

In another aspect of this embodiment, the hydrophobic substituent is agroup which, as a separate entity, is more soluble in octanol thanwater. For example, the octyl group (C₈H₁₇) is hydrophobic because itsparent alkane, octane, has greater solubility in octanol than in water.The hydrophobic substituent can be a saturated or unsaturated,substituted or unsubstituted hydrocarbon group. Such groups includesubstituted and unsubstituted, normal, branched or cyclic alkyl groupshaving at least four carbon atoms, substituted or unsubstitutedarylalkyl or heteroarylalkyl groups and substituted or unsubstitutedaryl or heteroaryl groups. Preferably, the hydrophobic substituentincludes an alkyl group of between about four and thirty carbons.Specific examples of suitable hydrophobic substituents include thefollowing alkyl groups n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,n-undecyl, n-dodecyl, n-tetradecyl, n-octadecyl, 2-ethylhexyl,3-propyl-6-methyl decyl, phenyl and combinations thereof. Other examplesof suitable hydrophobic substituents include haloalkyl groups of atleast six carbons (e.g., 10-halodecyl), hydroxyalkyl groups of at leastsix carbons (e.g., 11-hydroxyundecyl), and aralkyl groups (e.g.,benzyl).

In another aspect of this embodiment, R₂ is H or CH₃. In a furtheraspect of this embodiment, R₃ is ethoxy or isopropoxy.

In another embodiment, the invention is an erastin analog, such as acompound having formula Ia:

whereinR₁ is a hydrophobic or hydrophilic substituent, which is attached to oneor more positions of at least one carbon atom of the ring;R₃ is a C₂₋₈ alkoxy; oran enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the hydrophilic substituent isselected from alcohols, amines, nitro, carboxylic acids, carboxylates,hydroxy, amides, sulfamides, sulfonic acids, sulfonates, sulfates,esters, thiol esters, ethers, thiols, thiolates, thiol ethers,morpholino, fluoroaromatics, piperazines, piperadines, phosphonates, andsalts thereof, and combinations thereof. Preferably, the hydrophilicsubstituent is selected from NH₂, NO₂, NCOCH₃, and combinations thereof.

In another aspect of this embodiment, the hydrophobic substituent is agroup which, as a separate entity, is more soluble in octanol thanwater. For example, the octyl group (C₈H₁₇) is hydrophobic because itsparent alkane, octane, has greater solubility in octanol than in water.The hydrophobic substituent can be a saturated or unsaturated,substituted or unsubstituted hydrocarbon group. Such groups includesubstituted and unsubstituted, normal, branched or cyclic alkyl groupshaving at least four carbon atoms, substituted or unsubstitutedarylalkyl or heteroarylalkyl groups and substituted or unsubstitutedaryl or heteroaryl groups. Preferably, the hydrophobic substituentincludes an alkyl group of between about four and thirty carbons.Specific examples of suitable hydrophobic substituents include thefollowing alkyl groups n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,n-undecyl, n-dodecyl, n-tetradecyl, n-octadecyl, 2-ethylhexyl,3-propyl-6-methyl decyl, phenyl and combinations thereof. Other examplesof suitable hydrophobic substituents include haloalkyl groups of atleast six carbons (e.g., 10-halodecyl), hydroxyalkyl groups of at leastsix carbons (e.g., 11-hydroxyundecyl), and aralkyl groups (e.g.,benzyl).

In another aspect of this embodiment, R₃ is ethoxy or isopropoxy.

In a further aspect of this embodiment, the compound is an erastinanalog, such as a compound selected from:

an enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.

In another embodiment, the invention is an erastin analog, such as acompound of formula II:

whereinA is selected from the group consisting of C, N, and O;R₁ is a hydrophobic or hydrophilic substituent, which is attached to oneor more positions of at least one carbon atom of the ring with theproviso that when A is C, R₁ is not NH₂ or NO₂;R₂ is selected from H, C₁₋₈alkyl, C₁₋₈alkoxy, 3- to 8-memberedcarbocyclic or heterocyclic, aryl, heteroaryl, C₁₋₄aralkyl, residues ofglycolic acid, ethylene glycol/propylene glycol copolymers, carboxylate,ester, amide, carbohydrate, amino acid, alditol, OC(R₆)₂COOH,SC(R₆)₂COOH, NHCHR₆COOH, COR₇, CO₂R₇, sulfate, sulfonamide, sulfoxide,sulfonate, sulfone, thioalkyl, thioester, propylphthalimide, andthioether;R₃ is a C₂₋₈ alkoxy;R₄ is a hydrophilic substituent, which is attached to at least oneposition of A, except that when A is O, R₃ is nothing;R₆ is selected from H, C₁₋₈alkyl, carbocycle, aryl, heteroaryl,heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle, whereineach alkyl, carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, and alkylheterocycle may be optionally substituted withat least one substituent;R₇ is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl, aryl,carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, alkylheterocycle, and heteroaromatic may be optionallysubstituted with at least one substituent; oran enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the hydrophilic substituent isselected from the group consisting of alcohols, amines, nitro,carboxylic acids, carboxylates, hydroxy, amides, sulfamides, sulfonicacids, sulfonates, sulfates, esters, thiol esters, ethers, thiols,thiolates, thiol ethers, morpholino, fluoroaromatics, piperazines,piperadines, phosphonates, and salts thereof, and combinations thereof,with the proviso that when A is C, R₁ is not NH₂ or NO₂. Preferably, thehydrophilic substituent is NCOCH₃.

In another aspect of this embodiment, the hydrophobic substituent is agroup which, as a separate entity, is more soluble in octanol thanwater. For example, the octyl group (C₈H₁₇) is hydrophobic because itsparent alkane, octane, has greater solubility in octanol than in water.The hydrophobic substituent can be a saturated or unsaturated,substituted or unsubstituted hydrocarbon group. Such groups includesubstituted and unsubstituted, normal, branched or cyclic alkyl groupshaving at least four carbon atoms, substituted or unsubstitutedarylalkyl or heteroarylalkyl groups and substituted or unsubstitutedaryl or heteroaryl groups. Preferably, the hydrophobic substituentincludes an alkyl group of between about four and thirty carbons.Specific examples of suitable hydrophobic substituents include thefollowing alkyl groups n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,n-undecyl, n-dodecyl, n-tetradecyl, n-octadecyl, 2-ethylhexyl,3-propyl-6-methyl decyl, phenyl and combinations thereof. Other examplesof suitable hydrophobic substituents include haloalkyl groups of atleast six carbons (e.g., 10-halodecyl), hydroxyalkyl groups of at leastsix carbons (e.g., 11-hydroxyundecyl), and aralkyl groups (e.g.,benzyl).

In another aspect of this embodiment, R₂ is H or CH₃.

In a further aspect of this embodiment, R₃ is ethoxy or isopropoxy.

In a further aspect of this embodiment, A is C.

In another embodiment, the invention is an erastin analog, such as acompound selected from formula IIa, IIb, and IIc:

whereinR₁ is a hydrophobic or hydrophilic substituent, which is attached to oneor more positions of at least one carbon atom of the ring with theproviso that in formula IIa R₁ is not NH₂ or NO₂;R₂ is selected from H, C₁₋₈alkyl;R₃ is a C₂₋₈ alkoxy;R₄ and R₅, when present, are independently selected from the groupconsisting of H and an hydrophilic substituent; or an enantiomer,optical isomer, diastereomer, N-oxide, crystalline form, hydrate, orpharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the erastin analog is a compoundselected from:

an enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.

In another embodiment, the invention is a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound offormula I (including Ia) or II (including IIa, IIb, and IIc), includinge.g., one or more of compounds 1-20.

In another embodiment, the invention is a method of treating a conditionin a mammal, which comprises administering to the mammal atherapeutically effective amount of a compound of formula I (includingIa) or II (including IIa, IIb, and IIc), including e.g., one or more ofcompounds 1-20.

In the present invention, a “therapeutically effective amount” is anamount sufficient to effect beneficial or desired results. In terms oftreatment of a mammal, a “therapeutically effective amount” of acompound is an amount sufficient to treat, manage, palliate, ameliorate,or stabilize a condition, such as cancer, in the mammal. Atherapeutically effective amount can be administered in one or moredoses.

The therapeutically effective amount is generally determined by aphysician on a case-by-case basis and is within the skill of one in theart. Several factors are typically taken into account when determiningan appropriate dosage. These factors include age, sex and weight of thepatient, the condition being treated, the severity of the condition andthe form of the drug being administered.

Effective dosage forms, modes of administration, and dosage amounts maybe determined empirically, and making such determinations is within theskill of the art. It is understood by those skilled in the art that thedosage amount will vary with the route of administration, the rate ofexcretion, the duration of the treatment, the identity of any otherdrugs being administered, the age, size, and species of animal, and likefactors well known in the arts of medicine and veterinary medicine. Ingeneral, a suitable dose of a compound according to the invention willbe that amount of the compound, which is the lowest dose effective toproduce the desired effect. The effective dose of a compound maybeadministered as two, three, four, five, six or more sub-doses,administered separately at appropriate intervals throughout the day.

A compound of the present invention may be administered in any desiredand effective manner: as pharmaceutical compositions for oral ingestion,or for parenteral or other administration in any appropriate manner suchas intraperitoneal, subcutaneous, topical, intradermal, inhalation,intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous,intraarterial, intrathecal, or intralymphatic. Further, a compound ofthe present invention may be administered in conjunction with othertreatments. A compound or composition of the present invention maybeencapsulated or otherwise protected against gastric or other secretions,if desired.

While it is possible for a compound of the invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (“composition” or “pharmaceutical composition”). Thepharmaceutical compositions of the invention comprise one or morecompounds of the present invention as an active ingredient in admixturewith one or more pharmaceutically-acceptable carriers and, optionally,one or more other compounds, drugs, ingredients and/or materials.Regardless of the route of administration selected, the compounds of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington 's Pharmaceutical Sciences (Mack Publishing Co., Easton,Pa.).

Pharmaceutically acceptable carriers are well known in the art (see,e.g., Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton,Pa.) and The National Formulary (American Pharmaceutical Association,Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol,and sorbitol), starches, cellulose preparations, calcium phosphates(e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogenphosphate), sodium citrate, water, aqueous solutions (e.g., saline,sodium chloride injection, Ringer's injection, dextrose injection,dextrose and sodium chloride injection, lactated Ringer's injection),alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol),polyols (e.g., glycerol, propylene glycol, and polyethylene glycol),organic esters (e.g., ethyl oleate and tryglycerides), biodegradablepolymers (e.g., polylactide-polyglycolide, poly(orthoesters), andpoly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils(e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut),cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones,talc, silicylate, etc. Each pharmaceutically acceptable carrier used ina pharmaceutical composition of the invention must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not injurious to the subject. Carriers suitable for aselected dosage form and intended route of administration are well knownin the art, and acceptable carriers for a chosen dosage form and methodof administration can be determined using ordinary skill in the art.

The pharmaceutical compositions of the invention may, optionally,contain additional ingredients and/or materials commonly used inpharmaceutical compositions. These ingredients and materials are wellknown in the art and include (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, suchas carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, suchas glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,sodium starch glycolate, cross-linked sodium carboxymethyl cellulose andsodium carbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and glycerol monosterate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,and sodium lauryl sulfate; (10) suspending agents, such as ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth; (11) buffering agents; (12) excipients, such as lactose,milk sugars, polyethylene glycols, animal and vegetable fats, oils,waxes, paraffins, cocoa butter, starches, tragacanth, cellulosederivatives, polyethylene glycol, silicones, bentonites, silicic acid,talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, andpolyamide powder; (13) inert diluents, such as water or other solvents;(14) preservatives; (15) surface-active agents; (16) dispersing agents;(17) control-release or absorption-delaying agents, such ashydroxypropylmethyl cellulose, other polymer matrices, biodegradablepolymers, liposomes, microspheres, aluminum monosterate, gelatin, andwaxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21)emulsifying and suspending agents; (22), solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan; (23)propellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane; (24) antioxidants; (25) agentswhich render the formulation isotonic with the blood of the intendedrecipient, such as sugars and sodium chloride; (26) thickening agents;(27) coating materials, such as lecithin; and (28) sweetening,flavoring, coloring, perfuming and preservative agents. Each suchingredient or material must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Ingredients and materials suitable for aselected dosage form and intended route of administration are well knownin the art, and acceptable ingredients and materials for a chosen dosageform and method of administration may be determined using ordinary skillin the art.

Pharmaceutical compositions suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules, asolution or a suspension in an aqueous or non-aqueous liquid, anoil-in-water or water-in-oil liquid emulsion, an elixir or syrup, apastille, a bolus, an electuary or a paste. These formulations may beprepared by methods known in the art, e.g., by means of conventionalpan-coating, mixing, granulation or lyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared by mixing theactive ingredient(s) with one or more pharmaceutically-acceptablecarriers and, optionally, one or more fillers, extenders, binders,humectants, disintegrating agents, solution retarding agents, absorptionaccelerators, wetting agents, absorbents, lubricants, and/or coloringagents. Solid compositions of a similar type maybe employed as fillersin soft and hard-filled gelatin capsules using a suitable excipient. Atablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared using asuitable binder, lubricant, inert diluent, preservative, disintegrant,surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine. The tablets, and other solid dosageforms, such as dragees, capsules, pills and granules, may optionally bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.They may also be formulated so as to provide slow or controlled releaseof the active ingredient therein. They may be sterilized by, forexample, filtration through a bacteria-retaining filter. Thesecompositions may also optionally contain opacifying agents and may be ofa composition such that they release the active ingredient only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

Pharmaceutical compositions for rectal or vaginal administration may bepresented as a suppository, which maybe prepared by mixing one or moreactive ingredient(s) with one or more suitable nonirritating carrierswhich are solid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound. Pharmaceutical compositions which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing suchpharmaceutically-acceptable carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, drops and inhalants. The active compound may be mixed understerile conditions with a suitable pharmaceutically-acceptable carrier.The ointments, pastes, creams and gels may contain excipients. Powdersand sprays may contain excipients and propellants.

Pharmaceutical compositions suitable for parenteral administrationscomprise one or more compounds of the present invention in combinationwith one or more pharmaceutically-acceptable sterile isotonic aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain suitable antioxidants,buffers, solutes which render the formulation isotonic with the blood ofthe intended recipient, or suspending or thickening agents. Properfluidity can be maintained, for example, by the use of coatingmaterials, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. These compositions mayalso contain suitable adjuvants, such as wetting agents, emulsifyingagents and dispersing agents. It may also be desirable to includeisotonic agents. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption.

In some cases, in order to prolong the effect of a drug, it is desirableto slow its absorption from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility.

The rate of absorption of the drug then depends upon its rate ofdissolution which, in turn, may depend upon crystal size and crystallineform. Alternatively, delayed absorption of a parenterally-administereddrug may be accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms may be made by forming microencapsulematrices of the active ingredient in biodegradable polymers. Dependingon the ratio of the active ingredient to polymer, and the nature of theparticular polymer employed, the rate of active ingredient release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

With respect to the embodiment relating to a method of treating acondition in a mammal, the mammal is preferably a human. In certainother aspects of this embodiment, the condition is cancer. For example,the cancer may be leukemia, non-small cell lung carcinoma, colon cancer,CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer,breast cancer, and pancreatic cancer.

In certain other aspects, the method further comprises conjointlyadministering to the mammal an agent, such as a chemotherapeutic agent,that kills the cells through an apoptotic mechanism. In certainembodiments, the chemotherapeutic agent is selected from: anEGF-receptor antagonist, arsenic sulfide, adriamycin, cisplatin,carboplatin, cimetidine, caminomycin, mechlorethamine hydrochloride,pentamethylmelamine, thiotepa, teniposide, cyclophosphamide,chlorambucil, demethoxyhypocrellin A, melphalan, ifosfamide,trofosfamide, Treosulfan, podophyllotoxin or podophyllotoxinderivatives, etoposide phosphate, teniposide, etoposide, leurosidine,leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol,megestrol, methopterin, mitomycin C, ecteinascidin 743, busulfan,carmustine (BCNU), lomustine (CCNU), lovastatin,1-methyl-4-phenylpyridinium ion, semustine, staurosporine, streptozocin,phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate,thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin,cytarabine (ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine,doxorubicin hydrochloride, leucovorin, mycophenolic acid, daunorubicin,deferoxamine, floxuridine, doxifluridine, raltitrexed, idarubicin,epirubican, pirarubican, zorubicin, mitoxantrone, bleomycin sulfate,actinomycin D, safracins, saframycins, quinocarcins, discodermolides,vincristine, vinblastine, vinorelbine tartrate, vertoporfin, paclitaxel,tamoxifen, raloxifene, tiazofuran, thioguanine, ribavirin, EICAR,estramustine, estramustine phosphate sodium, flutamide, bicalutamide,buserelin, leuprolide, pteridines, enediynes, levamisole, aflacon,interferon, interleukins, aldesleukin, filgrastim, sargramostim,rituximab, BCG, tretinoin, betamethosone, gemcitabine hydrochloride,verapamil, VP-16, altretamine, thapsigargin, oxaliplatin, iproplatin,tetraplatin, lobaplatin, DCP, PLD-147, JM118, JM216, JM335, satraplatin,docetaxel, deoxygenated paclitaxel, TL-139, 5′-nor-anhydrovinblastine(hereinafter: 5′-nor-vinblastine), camptothecin, irinotecan (Camptosar,CPT-11), topotecan (Hycamptin), BAY 38-3441, 9-nitrocamptothecin(Orethecin, rubitecan), exatecan (DX-8951), lurtotecan (GI-147211C),gimatecan, homocamptothecins diflomotecan (BN-80915) and9-aminocamptothecin (IDEC-13′), SN-38, ST1481, karanitecin (BNP1350),indolocarbazoles (e.g., NB-506), protoberberines, intoplicines,idenoisoquinolones, benzo-phenazines, NB-506, or combinations thereof.

In another embodiment, the invention is a method of treating a conditionin a mammal. This method comprises administering to the mammal atherapeutically effective amount of a pharmaceutical compositioncomprising a compound of formula I (including Ia) or II (including IIa,IIb, and IIc), including e.g., one or more of compounds 1-20.

In certain aspects of this embodiment, the mammal is preferably a human.In certain other aspects of this embodiment, the condition is cancer.For example, the cancer may be leukemia, non-small cell lung carcinoma,colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer,prostate cancer, breast cancer, and pancreatic cancer.

In certain other aspects, the method further comprises conjointlyadministering to the mammal an agent, such as a chemotherapeutic agent,that kills the cells through an apoptotic mechanism. In certainembodiments, the chemotherapeutic agent is selected from: anEGF-receptor antagonist, arsenic sulfide, adriamycin, cisplatin,carboplatin, cimetidine, caminomycin, mechlorethamine hydrochloride,pentamethylmelamine, thiotepa, teniposide, cyclophosphamide,chlorambucil, demethoxyhypocrellin A, melphalan, ifosfamide,trofosfamide, Treosulfan, podophyllotoxin or podophyllotoxinderivatives, etoposide phosphate, teniposide, etoposide, leurosidine,leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol,megestrol, methopterin, mitomycin C, ecteinascidin 743, busulfan,carmustine (BCNU), lomustine (CCNU), lovastatin,1-methyl-4-phenylpyridinium ion, semustine, staurosporine, streptozocin,phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate,thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin,cytarabine (ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine,doxorubicin hydrochloride, leucovorin, mycophenolic acid, daunorubicin,deferoxamine, floxuridine, doxifluridine, raltitrexed, idarubicin,epirubican, pirarubican, zorubicin, mitoxantrone, bleomycin sulfate,actinomycin D, safracins, saframycins, quinocarcins, discodermolides,vincristine, vinblastine, vinorelbine tartrate, vertoporfin, paclitaxel,tamoxifen, raloxifene, tiazofuran, thioguanine, ribavirin, EICAR,estramustine, estramustine phosphate sodium, flutamide, bicalutamide,buserelin, leuprolide, pteridines, enediynes, levamisole, aflacon,interferon, interleukins, aldesleukin, filgrastim, sargramostim,rituximab, BCG, tretinoin, betamethosone, gemcitabine hydrochloride,verapamil, VP-16, altretamine, thapsigargin, oxaliplatin, iproplatin,tetraplatin, lobaplatin, DCP, PLD-147, JM118, JM216, JM335, satraplatin,docetaxel, deoxygenated paclitaxel, TL-139, 5′-nor-anhydrovinblastine(hereinafter: 5′-nor-vinblastine), camptothecin, irinotecan (Camptosar,CPT-11), topotecan (Hycamptin), BAY 38-3441, 9-nitrocamptothecin(Orethecin, rubitecan), exatecan (DX-8951), lurtotecan (GI-147211C),gimatecan, homocamptothecins diflomotecan (BN-80915) and9-aminocamptothecin (IDEC-13′), SN-38, ST1481, karanitecin (BNP1350),indolocarbazoles (e.g., NB-506), protoberberines, intoplicines,idenoisoquinolones, benzo-phenazines, NB-506, or combinations thereof.

In another embodiment, the invention is an erastin analog, such as acompound selected from:

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.

One aspect of this embodiment is a pharmaceutical composition comprisinga pharmaceutically acceptable carrier and a compound selected fromcompounds 22-24, 34, or 40, or a combination thereof, or an enantiomer,optical isomer, diastereomer, N-oxide, crystalline form, hydrate, orpharmaceutically acceptable salt thereof.

Another aspect of this embodiment, is a method of treating a conditionin a mammal. In this method, the mammal is administered atherapeutically effective amount of a compound according to the presentinvention, such as, e.g., compounds 22-24, 34, or 40, or a combinationthereof, or an enantiomer, optical isomer, diastereomer, N-oxide,crystalline form, hydrate, or pharmaceutically acceptable salt thereof.In this embodiment, the mammal is preferably a human. In certain otheraspects of this embodiment, the condition is cancer. For example, thecancer may be leukemia, non-small cell lung carcinoma, colon cancer, CNScancer, melanoma, ovarian cancer, renal cancer, prostate cancer, breastcancer, and pancreatic cancer.

In certain other aspects of this embodiment, the method furthercomprises conjointly administering to the mammal an agent, such as achemotherapeutic agent, that kills the cells through an apoptoticmechanism. In certain embodiments, the chemotherapeutic agent isselected from: an EGF-receptor antagonist, arsenic sulfide, adriamycin,cisplatin, carboplatin, cimetidine, caminomycin, mechlorethaminehydrochloride, pentamethylmelamine, thiotepa, teniposide,cyclophosphamide, chlorambucil, demethoxyhypocrellin A, melphalan,ifosfamide, trofosfamide, Treosulfan, podophyllotoxin or podophyllotoxinderivatives, etoposide phosphate, teniposide, etoposide, leurosidine,leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol,megestrol, methopterin, mitomycin C, ecteinascidin 743, busulfan,carmustine (BCNU), lomustine (CCNU), lovastatin,1-methyl-4-phenylpyridinium ion, semustine, staurosporine, streptozocin,phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate,thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin,cytarabine (ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine,doxorubicin hydrochloride, leucovorin, mycophenolic acid, daunorubicin,deferoxamine, floxuridine, doxifluridine, raltitrexed, idarubicin,epirubican, pirarubican, zorubicin, mitoxantrone, bleomycin sulfate,actinomycin D, safracins, saframycins, quinocarcins, discodermolides,vincristine, vinblastine, vinorelbine tartrate, vertoporfin, paclitaxel,tamoxifen, raloxifene, tiazofuran, thioguanine, ribavirin, EICAR,estramustine, estramustine phosphate sodium, flutamide, bicalutamide,buserelin, leuprolide, pteridines, enediynes, levamisole, aflacon,interferon, interleukins, aldesleukin, filgrastim, sargramostim,rituximab, BCG, tretinoin, betamethosone, gemcitabine hydrochloride,verapamil, VP-16, altretamine, thapsigargin, oxaliplatin, iproplatin,tetraplatin, lobaplatin, DCP, PLD-147, JM118, JM216, JM335, satraplatin,docetaxel, deoxygenated paclitaxel, TL-139, 5′-nor-anhydrovinblastine(hereinafter: 5′-nor-vinblastine), camptothecin, irinotecan (Camptosar,CPT-11), topotecan (Hycamptin), BAY 38-3441, 9-nitrocamptothecin(Orethecin, rubitecan), exatecan (DX-8951), lurtotecan (GI-147211C),gimatecan, homocamptothecins diflomotecan (BN-80915) and9-aminocamptothecin (IDEC-13′), SN-38, ST1481, karanitecin (BNP1350),indolocarbazoles (e.g., NB-506), protoberberines, intoplicines,idenoisoquinolones, benzo-phenazines, NB-506, or combinations thereof.

In another embodiment, the invention is a method of treating a conditionin a mammal. This method comprises administering to the mammal atherapeutically effective amount of a pharmaceutical compositioncomprising compounds 22-24, 34, or 40, or a combination thereof, or anenantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.

In certain aspects of this embodiment, the mammal is preferably a human.In certain other aspects of this embodiment, the condition is cancer.For example, the cancer may be leukemia, non-small cell lung carcinoma,colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer,prostate cancer, breast cancer, and pancreatic cancer.

In certain other aspects, the method further comprises conjointlyadministering to the mammal an agent, such as a chemotherapeutic agent,that kills the cells through an apoptotic mechanism. In certainembodiments, the chemotherapeutic agent is selected from: anEGF-receptor antagonist, arsenic sulfide, adriamycin, cisplatin,carboplatin, cimetidine, caminomycin, mechlorethamine hydrochloride,pentamethylmelamine, thiotepa, teniposide, cyclophosphamide,chlorambucil, demethoxyhypocrellin A, melphalan, ifosfamide,trofosfamide, Treosulfan, podophyllotoxin or podophyllotoxinderivatives, etoposide phosphate, teniposide, etoposide, leurosidine,leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol,megestrol, methopterin, mitomycin C, ecteinascidin 743, busulfan,carmustine (BCNU), lomustine (CCNU), lovastatin,1-methyl-4-phenylpyridinium ion, semustine, staurosporine, streptozocin,phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate,thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin,cytarabine (ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine,doxorubicin hydrochloride, leucovorin, mycophenolic acid, daunorubicin,deferoxamine, floxuridine, doxifluridine, raltitrexed, idarubicin,epirubican, pirarubican, zorubicin, mitoxantrone, bleomycin sulfate,actinomycin D, safracins, saframycins, quinocarcins, discodermolides,vincristine, vinblastine, vinorelbine tartrate, vertoporfin, paclitaxel,tamoxifen, raloxifene, tiazofuran, thioguanine, ribavirin, EICAR,estramustine, estramustine phosphate sodium, flutamide, bicalutamide,buserelin, leuprolide, pteridines, enediynes, levamisole, aflacon,interferon, interleukins, aldesleukin, filgrastim, sargramostim,rituximab, BCG, tretinoin, betamethosone, gemcitabine hydrochloride,verapamil, VP-16, altretamine, thapsigargin, oxaliplatin, iproplatin,tetraplatin, lobaplatin, DCP, PLD-147, JM118, JM216, JM335, satraplatin,docetaxel, deoxygenated paclitaxel, TL-139, 5′-nor-anhydrovinblastine(hereinafter: 5′-nor-vinblastine), camptothecin, irinotecan (Camptosar,CPT-11), topotecan (Hycamptin), BAY 38-3441, 9-nitrocamptothecin(Orethecin, rubitecan), exatecan (DX-8951), lurtotecan (GI-147211C),gimatecan, homocamptothecins diflomotecan (BN-80915) and9-aminocamptothecin (IDEC-13′), SN-38, ST1481, karanitecin (BNP1350),indolocarbazoles (e.g., NB-506), protoberberines, intoplicines,idenoisoquinolones, benzo-phenazines, NB-506, or combinations thereof.

It is contemplated that all embodiments of the invention can be combinedwith one or more other embodiments, even those described under differentaspects of the invention. In the foregoing embodiments, the followingdefinitions apply.

As used herein, the term “acyl” has its art-recognized meaning andrefers to a group represented by the general formula hydrocarbylC(O)—,preferably alkylC(O)—.

As used herein, the term “acylamino” has its art-recognized meaning andrefers to an amino group substituted with an acyl group and may berepresented, for example, by the formula hydrocarbylC(O)NH—.

The term “alkoxy” refers to an alkyl group, preferably a lower alkylgroup, having an oxygen attached thereto. Representative alkoxy groupsinclude methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Such substituents may occur onone or more carbons that are included or not included in one or moredouble bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed below, except where stability isprohibitive. For example, substitution of alkenyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer, such as from 1 to 8. Likewise,preferred cycloalkyls have from 3-10 carbon atoms in their ringstructure, such as 3-8, including 5, 6 or 7 carbons in the ringstructure.

Moreover, unless otherwise indicated, the term “alkyl” (or “loweralkyl”) as used throughout the specification, examples, and claims isintended to include both “unsubstituted alkyls” and “substitutedalkyls”, the latter of which refers to alkyl moieties havingsubstituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, ahalogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl,a formyl, or an acyl), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, aphosphonate, a phosphinate, an amino, an amido, an amidine, an imine, acyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, asulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, anaralkyl, an aromatic, or heteroaromatic moiety. It will be understood bythose skilled in the art that the moieties substituted on thehydrocarbon chain can themselves be substituted, if appropriate. Forinstance, the substituents of a substituted alkyl may includesubstituted and unsubstituted forms of amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Exemplary substitutedalkyls are described below. Cycloalkyls can be further substituted withalkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substitutedalkyls, —CF₃, —CN, and the like.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, alkyl, alkenyl, or alkoxy is meant to include groups that containfrom x to y carbons in the chain. For example, the term “C_(x-y)alkyl”refers to substituted or unsubstituted saturated hydrocarbon groups,including straight-chain alkyl and branched-chain alkyl groups thatcontain from x to y carbons in the chain, including haloalkyl groupssuch as trifluoromethyl and 2,2,2-tirfluoroethyl, etc. The terms“C_(2-y)alkenyl” and “C_(2-y)alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS-.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls”, the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Such substituents may occur onone or more carbons that are included or not included in one or moretriple bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed above, except where stability isprohibitive. For example, substitution of alkynyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “amide”, as used herein, refers to a group

wherein R⁷ and R⁸ each independently represent a hydrogen or hydrocarbylgroup, or R⁷ and R⁸ taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein R⁷, R⁸, and R⁸′ each independently represent a hydrogen or ahydrocarbyl group, or R⁷ and R⁸ taken together with the N atom to whichthey are attached complete a heterocycle having from 4 to 8 atoms in thering structure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein includes substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 3- to 8-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein R⁷ and R⁸ independently represent hydrogen or a hydrocarbylgroup.

The terms “carbocycle”, “carbocyclyl”, and “carbocyclic”, as usedherein, refer to a non-aromatic saturated or unsaturated ring in whicheach atom of the ring is carbon. Preferably a carbocycle ring containsfrom 3 to 10 atoms, more preferably from 3 to 8 atoms, including 5 to 7atoms, such as for example, 6 atoms.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “ester”, as used herein, refers to a group —C(O)OR⁷ wherein R⁷represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” are used interchangeably herein and meanhalogen and include chloro, fluoro, bromo, and iodo.

The term “heteroaryl” includes substituted or unsubstituted aromaticsingle ring structures, preferably 3- to 8-membered rings, morepreferably 5- to 7-membered rings, even more preferably 5- to 6-memberedrings, whose ring structures include at least one heteroatom, preferablyone to four heteroatoms, more preferably one or two heteroatoms. Theterm “heteroaryl” also includes polycyclic ring systems having two ormore cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The terms “heterocyclyl”, “heterocycle”, “heterocyclic”, and the likerefer to substituted or unsubstituted non-aromatic ring structures,preferably 3- to 10-membered rings, more preferably 3- to 8-memberedrings, whose ring structures include at least one heteroatom, preferablyone to four heteroatoms, more preferably one or two heteroatoms. Theterms “heterocyclyl,” “heterocyclic,” and the like also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is heterocyclic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heterocyclyl groups include, for example, piperidine,piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groupswhere there are ten or fewer non-hydrogen atoms in the substituent,preferably eight or fewer, such as for example, from about 2 to 8 carbonatoms, including less than 6 carbon atoms. A “lower alkyl”, for example,refers to an alkyl group that contains ten or fewer carbon atoms,preferably eight or fewer. In certain embodiments, acyl, alkyl, alkenyl,alkynyl, or alkoxy substituents defined herein are respectively loweracyl, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy,whether they appear alone or in combination with other substituents,such as in the recitations hydroxyalkyl and aralkyl (in which case, forexample, the atoms within the aryl group are not counted when countingthe carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 3 to 8, such as for example, 5 to 7.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with the permitted valence ofthe substituted atom and the substituent, and that the substitutionresults in a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate.

As used herein, the term “substituent,” particularly with respect to R₆and R₇ in formulae I and II, means H, cyano, oxo, nitro, acyl,acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate, ester,ether, carboxylic acid, thio, thioalkyl, thioester, thioether, C₁₋₈alkyl, C₁₋₈alkoxy, C₁₋₈alkenyl, C₁₋₈aralkyl, 3- to 8-memberedcarbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl, or 3-to 8-membered heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate,sulfone, alkylsulfonyl, and arylsulfonyl.

Unless specifically stated as “unsubstituted,” references to chemicalmoieties herein are understood to include substituted variants. Forexample, reference to an “aryl” group or moiety implicitly includes bothsubstituted and unsubstituted variants.

The term “sulfate” is art-recognized and refers to the group —OSO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

wherein R⁷ and R⁸ independently represents hydrogen or hydrocarbyl.

The term “sulfoxide” is art-recognized and refers to the group —S(O)—R⁷,wherein R⁷ represents a hydrocarbyl.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)₂—R⁷,wherein R⁷ represents a hydrocarbyl.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR⁷ or—SC(O)R⁷ wherein R⁷ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the generalformula

wherein R⁷ and R⁸ independently represent hydrogen or a hydrocarbyl.

The following examples are provided to further illustrate the methodsand compositions of the present invention. These examples areillustrative only and are not intended to limit the scope of theinvention in any way.

EXAMPLES Engineered Human Tumor Cells

Primary human cells can be converted into tumorigenic cells byintroduction of vectors expressing hTERT, oncogenic RAS, and otherproteins that disrupt the function of p53, RB, and PP2A⁵⁵⁻⁶⁰. Suchengineered human tumorigenic cells and their precursors (FIG. 1), werecreated from primary human foreskin fibroblasts. Characteristics ofthese cells reported previously include doubling time, resistance tosenescence and crisis in culture, response to irradiation, ability togrow in an anchorage-independent fashion, and ability to form tumors inmice^(56, 57, 60). These cells were used to discover RAS-selectivelethal compounds, including a compound named erastin.

Cell Culture and Western Blotting:

BJ-TERT/LT/ST/RAS^(V12) cells were cultured as described (Dolma, S.,Lessnick, S. L., Hahn, W. C. & Stockwell, B. R. Identification ofgenotype-selective antitumor agents using synthetic lethal chemicalscreening in engineered human tumor cells. Cancer Cell. 3: 285-96(2003). Other cell lines were grown according to specifications of theAmerican Type Culture Collection. For BRAF knockdown, A673 cells wereinfected with pSIRIPP-derived retroviruses (Sage, J., Miller, A. L.,Perez-Mancera, P. A., Wysocki, J. M. & Jacks, T. Acute mutation ofretinoblastoma gene function is sufficient for cell cycle re-entry.Nature. 424: 223-8 (2003), expressing short-hairpin RNAs against eitherBRAF or luciferase, and were selected in 2 μg/ml puromycin to removeuninfected cells. The sequences of the cloned oligonucleotides were asfollows: BRAF:5′-GAT CCC CGT GTT GGA GAA TGT TCC ACT TCA AGA GAG TGG AACATT CTC CAA CAC TTT TTG GAA A-3′ (SEQ ID NO:28); Luciferase: 5′-GAT CCCCCT TAC GCT GAG TAC TTC GAT TCA AGA GAT CGA AGT ACT CAG CGT MG TTT TTGGAA A-3′ (SEQ ID NO:29).

For western blots, medium was aspirated, and each dish was washed twicewith 10 mL of ice-cold PBS. Cells were lysed with 200 μL of buffer (50mM HEPES, 40 mM NaCl, 2 mM EDTA, 0.5% Triton-X, 1.5 mM sodiumorthovanadate, 50 mM NaF, 10 mM Na Pyrophosphate, 10 mM NaB-glycerophosphate and protease inhibitor tablet, pH 7.4). Samples wereseparated using SDS-PAGE, transferred to a PVDF membrane, blocked for 1hour at room temperature in Licor Odyssey Blocking Buffer and incubatedwith the necessary primary and secondary antibodies: anti-VDAC1 (Abcam,#ab3434), anti-VDAC1 (Calbiochem, #529534), anti-VDAC2 (Abcam,#ab22170), anti-eIF4E (Santa Cruz Biotechnology, #sc-9976),anti-α-Tubulin (Sigma, #T6199), anti-actin (Santa Cruz Biotechnology,#1616R), IRDye 800 goat anti-rabbit antibody (Rockland Immunochemicals,#611-132-122), Alexa Fluor 680 goat anti-, mouse (Molecular Probes,#A21058), PathScan Multiplex Western Cocktail I Kit (Cell SignalingTechnology), anti-PARP (Abcam, #ab105). Membranes were scanned using theLicor Odyssey™ Imaging System.

PARP Cleavage and Cytochrome C Release:

BJ-TERT/LT/ST/RAS^(V12) cells were seeded in polystyrene 100×20 mmdishes (Falcon/#353003) in 10 ml of media. Three million cells wereseeded in each dish. After overnight incubation at 37° C. with 5% CO₂,BJ-TERT/LT/ST/RAS^(V12) cells were treated with nothing, staurosporine(1 μM) for 6 h, camptothecin (1 μM) for 18 h or erastin (20 μg/mL) for6, 10, 12, 12.5, 13, 14, 18 or 26 h, and prepared for western blotting.

For the cytochrome c release assay, cells were washed with 10 mL ofice-cold PBS, suspended in 120 μL of buffer (300 mM sucrose, 0.1% BSA,10 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EGTA, 1 mM EDTA, 1 mMdithiothreitol, 2 mM phenylmethanesulfonyl fluoride and 1 proteaseinhibitor tablet (Roche)) and incubated on ice for 15 min. Cells werelysed by passing them through a 25-gauge needle (five strokes). Celllysates were centrifuged at 1850 rpm for 5 min at 4° C. to remove thenuclear fraction. Mitochondria were removed from the soluble cytosolicfraction by pelleting at 10000 rpm. Supernatant and mitochondrialpellets were solubilized in SDS-PAGE loading buffer and analyzed bywestern blotting.

Oxidative Species Detection:

2′7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA, Molecular Probes)was used to measure oxidative species by flow cytometry. Non-fluorescentH₂DCF-DA is cleaved by endogenous esterases and then is oxidized togenerate fluorescent dichlorofluorescein (DCF). BJ-TERT/LT/ST/RAS^(V12)and BJ-TERT cells were seeded at 3×10⁵ cells per dish in 60-mm dishesand allowed to grow overnight. Cells were treated with 4.6 μM erastinfor 2, 4, 6, 8, 10 and 12 h. For each time point, controls weremaintained for untreated cells and also for positive control (treateddirectly with 500 μM hydrogen peroxide for five minutes). Cells wereincubated with 10 μM of H₂DCF-DA for 10 minutes, harvested bytrypsinization, washed twice with cold PBS, resuspended in 100 μl of PBSand incubated with 5 μl of 50-μg/ml propidium iodide for 10 minutes. 400μl of PBS was added and the solution analyzed by flow cytometry(FACSCalibur-Becton-Dickinson). FL1-H indicates DCF fluorescence unitsdetected.

VDAC Chemi-Proteomic Identification:

Cultures of BJ-TERT and BJ-TERT/LT/ST/RAS^(V12) cells (ten 150 mmplates) were washed with PBS, lysed in 25 mMhydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) pH 7.5, 150 mMNaCl, 1% NP40, 10 mM MgCl₂, 1 mM ethylene-diamine tetra acetate (EDTA),10% glycerol, 1 mM dithiothreitol (DTT) and protease inhibitor cocktail.Protein concentration was determined using a Bradford colorimetricassay.

Erastin A3 and B2 were dissolved in DMSO at 10 mg/ml. 100 μL ofAffiGel-10 was washed and resuspended in 400 μl DMSO. 10 μL of compoundand 3 μL of 1:100 dilution of triethylamine in DMSO were added. Thesuspension was incubated at RT for 12 h, washed (1 mL/wash) 5×DMSO,3×PBS, resuspended in 3 M ethanolamine in PBS, incubated 1 h at RT,washed 5×PBS, diluted 1:1 in PBS, washed with HEGN binding buffer (0.1MKCl, 20 mM HEPES, pH 7.6, 0.1 mM EDTA, 10% glycerol, 0.1% NP40, 1 mMDTT, 0.25 mM PMSF) and incubated with 1 ml cell lysate (2 mg/ml) for 1.5at 4° C., washed with HEGN binding buffer, 3× with HEGN high salt buffer(0.35M KCl, 20 mM HEPES, pH 7.6, 0.1 mM EDTA, 10% glycerol, 0.1% NP40, 1mM DTT, 0.25 mM PMSF), 1× with HEGN binding buffer, and eluted 2× with50 μL HEGN elution buffer (HEGN binding buffer, 0.8% N-lauroylsarcosine) 15 min each; proteins from the supernatant were precipitatedwith 400 μL ethanol, sedimented by centrifugation (14,000 rpm) anddigested as described (Zheng, Y. et al. Essential role of thevoltage-dependent anion channel (VDAC) in mitochondrial permeabilitytransition pore opening and cytochrome c release induced by arsenictrioxide. Oncogene. 23: 1239-47 (2004). Reverse-phase-HPLC was performedusing a nano LC system from Dionex: a 75 μm×150 mm column, a Famosautosampler, a Switchos II system and an UltiMate binary pumping module.Samples were analyzed using both a 4700 Proteomics AnalyzerMALDI-TOF/TOF (TOF/TOF; Applied Biosystems) and a Q Trap (AB/MDS Sciez)and the peptide level data were combined. To construct the databasesused for protein identification, the following steps were performed: TheNCBInr protein sequence FASTA file was downloaded, the gi numbers wereupdated, and the missing or incorrectly annotated taxonomies were fixedby referencing them to the NCBI taxonomy index (index of gi number vs.species). The human subset of proteins in the database was extractedinto a separate database (HumanNR). All protein sequences in HumanNRwere matched to the corresponding protein in RefSeq using BLAST(Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J.Basic local alignment search tool. J Mol Biol. 215: 403-10 (1990). MS/MSdata obtained from the TOF/TOF and Q TRAP were searched using Mascot(Matrix Sciences, London, UK). All searches were performed againsteither the corrected NCBInr protein sequence database or the HumanNRdatabase. GPS Explorer (Applied Biosystems) was used for submitting dataacquired from the TOF/TOF for database searching. The Mascot-basedsearch was performed using the default settings for the specificinstrument type as supplied by Matrix Science, except that ions withscores below 10 were excluded from the results.

The spectra of the peptides identified in the automatic data analysiswere manually inspected for the quality of the corresponding spectra andconsistency with the obtained results. Only high quality spectra andresults with a peptide score of 20 or higher were accepted and used forthe identification of proteins.

Transmission Electron Microscopy of BJ-Tert/LT/ST/RAS^(V12) CellsTreated with Erastin:

Cells were fixed 24 h with 2.5% glutaraldehyde in 0.1 M Sorenson'sbuffer (0.1 M H2PO4, 0.1 M HPO4, pH 7.2) for at least one hour, treatedwith 1% OsO4 in 0.1 M Sorenson's buffer for 1 h. Enblock staining used1% tannic acid. After dehydration through an ethanol series, cells wereembedded in Lx-112 (Ladd Research Industries, Inc) and Embed-812 (EMS,Fort Washington Pa.). Thin sections were cut on an MT-7000ultramicrotome, stained with 1% uranyl acetate and 0.4% lead citrate andexamined under a JEOL JEM-1200 EXII electron microscope. Pictures weretaken on an ORCA-HR digital camera (Hamamatsu) at ˜20,000-foldmagnification, and measurements were made using the AMT Image CaptureEngine.

ATP Detection:

Cells were seeded in polystyrene 100×20 mm dishes (Falcon/#353003) in 10mL of media. Three million cells were seeded in each dish forstaurosporine (STS) and erastin treatment. Two million cells were seededin each dish for no treatment and hydrogen peroxide treatment. Cellswere treated with staurosporine (1 μM) for 12 hours, with H₂O₂ (16 mM)for 1.5 hours and erastin (20 μg/mL) for 12 hours. After the incubation,we counted cells using a CiCell analyzer (Beckman). 100,000 cells fromeach treatment were collected and washed twice in Hank's Salt Solution(10 ml). Next, we suspended cells in 200 μl of Nucleotide ReleasingBuffer (BioVision/K254-200 ApoSensor Cell Viability Assay Kit) andincubated at room temperature for 10 minutes. To 10 μl of the abovesample, we added 1 μl of ATP Minitoring Enzyme (BioVision/K254-200ApoSensor Cell Viability Assay Kit) and read the ATP levels of thesamples using a luminometer.

Knockdown using Lentiviral shRNAs:

VDACs, KRAS and BRAF were knocked down in HT1080, Calu-1, and A673cells, respectively, using short hairpin RNA lentiviral vectors. On day1, 293T cells were seeded in 10 cm tissue culture dishes (2×10⁶cells/dish). On day 2, shRNA-plasmid construct (pLKO.1 vector) and thepDelta.8.9 and pVSV-G helper plasmids were co-transfected into the 293Tcells using FuGENE® 6 Transfection Reagent. On day 3, the medium waschanged. On day 4, the supernatant, containing virus, was transferred toHT1080 cells in 10 cm tissue culture dishes (1×10⁶ cells/dish). On day5, cells were transferred to 175 cm² flasks and medium was supplementedwith puromycin. On days 6 and 7, medium was changed and supplementedwith puromycin. On day 8, samples were harvested for Western Blot andquantitative RT-PCR, or reseeded in 6-well format (5×10⁵ cells/well), induplicate, and treated with erastin dilutions. For BRAF knockdown, A673cells were infected with pSIRIPP-derived retroviruses expressingshort-hairpin RNAs against either BRAF or luciferase, and were selectedin 1.5 μg/ml puromycin to remove uninfected cells. All cells werecultured at 37° C., 5% CO₂, in growth media as recommended by ATCC.(Sage, J., Miller, A. L., Perez-Mancera, P. A., Wysocki, J. M. & Jacks,T. Acute mutation of retinoblastoma gene function is sufficient for cellcycle re-entry. Nature 424, 223-8 (2003).)

ATP levels were measured in BJERL cells treated with nothing (NT),treated with 1 μM of Staurosporine (STS) for 12 hours, with 16 mM ofhydrogen peroxide for 1.5 hours and 20 μg/mL of erastin for 12 hours.ATP levels were normalized by number of viable cells.

VDAC3 shRNAs Induce Isoform-Selective Knockdown in HT1080 Cells.Briefly, the assay was carried out as follows: Day 1, 293T cells seededin 10 cm tissue culture dishes (2×10⁶ cells/dish); Day 2, shRNA-plasmidconstruct (pLKO.1 vector) introduced to cells using FuGene transfectionreagent; Day 3, medium changed; Day 4, supernatant transferred to HT1080cells in 10 cm tissue culture dishes (1×10⁵ cells/dish); Day 5, cellstransferred to 175 cm² flasks, medium supplemented with puromycin; Days6 and 7, medium changed and supplemented with puromycin; Day 8, samplesharvested for qRT-PCR. Unique shRNAs (V3.B1, V3.B2, V3.B3, V3.B5, V3.B5,V3.B6), control construct (GFP).

VDAC1 shRNAs Induce Isoform-Selective Knockdown in HT1080 Cells.Briefly, the assay was carried out as follows: Day 1, 293T cells seededin 10 cm tissue culture dishes (2×10⁶ cells/dish); Day 2, shRNA-plasmidconstruct (pLKO.1 vector) introduced to cells using FuGene transfectionreagent; Day 3, medium changed; Day 4, supernatant transferred to HT1080cells in 10 cm tissue culture dishes (1×10⁶ cells/dish); Day 5, cellstransferred to 175 cm² flasks, medium supplemented with puromycin; Days6 and 7, medium changed and supplemented with puromycin; Day 8, samplesharvested for qRT-PCR. Unique shRNAs (V1.161, V1.279, V1.396, V1.607,V1.921), control construct (GFP).

Overexpression of VDAC3 Using Lentiviral Constructs:

VDAC3 was overexpressed in BJ-TERT cells, respectively, using a humanORF clone (Invitrogen) recombined into the pLENTi6/V5-DEST lentiviralvector (Invitrogen). On day 1, 293T cells were seeded in 10 cm tissueculture dishes (2×10⁶ cells/dish). On day 2, the VDAC3 construct and thepDelta8.9 and pVSV-G helper plasmids were co-transfected into the 293Tcells using FuGENE® 6 Transfection Reagent (Roche). On day 3, the mediumwas changed. On day 4, the supernatant, containing virus, wastransferred to BJ-TERT cells in 10 cm tissue culture dishes (1×10⁶cells/dish). On day 5, cells were transferred to 175 cm² flasks andmedium was supplemented with Blasticidin (5 μg/mL). On days 6 and 7,medium was changed and again supplemented with Blasticidin. These cellswere maintained in selection medium for 12 days before samples wereharvested for Western Blot and quantitative RT-PCR, or reseeded in6-well format (5×10⁵ cells/well), in duplicate, and treated with erastindilutions.

Reverse Transcription and Quantitative PCR:

Total RNA was isolated from cells using RNeasy Mini Kit (QIAGEN).Reverse Transcription was performed on 2 μg of isolated RNA using TaqmanReverse Transcription Reagents (Applied Biosystems). The ABI Prism 7300was then used for Quantitative PCR. 20 ng of cDNA product was mixed withPower SYBR Green PCR Master Mix (Applied Biosystems) and the appropriateforward/reverse primer set. Relative mRNA expression levels werequantified with Applied Biosystems Sequence Detection Software v1.3.1.

NADH Oxidation Assay:

NADH oxidation in the mitochondria was measured by resuspendingmitochondria isolated from yeast in R-buffer (0.65 M sucrose, 10 mMHEPES (pH 7.5), 10 mM KH₂PO₄, 5 mM KCl, 5 mM, MgCl₂) to a finalconcentration of 100 μg/ml. Mitochondrial concentration was measured bydissolving mitochondria in 0.6% SDS and reading absorbance at 280 nm.The mitochondrial suspension was then incubated with 25 μM NADH, and theabsorbance at 340 nm monitored over a 15 minute period. The assay wasrepeated in the presence of erastin. To assess mitochondrial intactness,a parallel assay was run in which mitochondria were hypotonicallyshocked prior to addition of NADH. The mitochondrial pellet wasresuspended in dH₂O and incubated on ice for 3 minutes to disrupt theouter mitochondrial membrane. 2× R-buffer was then added to restorenormal osmotic conditions.

Cell Viability Assays:

Trypan Blue exclusion: Cells were trypsinized, pelleted, resuspended in1 mL growth media. Trypan Blue exclusion analysis was performed usingthe Vi-CELL™ Series Cell Viability Analyzer 1.01 (Beckman Coulter).

Alamar Blue metabolism: 10% Alamar Blue was added to assay plates, whichwere then incubated for an additional 16 hours. Red fluorescence,resulting from reduction of Alamar Blue, was detected on a Victor3platereader (ex: 530, em: 590).

VDAC2 Binding Assay:

VDAC2 protein was isolated from E. coli using a modified version of theprotocol originally described by Koppel et al. Bacterial cultures weregrown in LB containing 50 mg/L ampicillin to an OD of 0.6, and inducedusing 0.4 μM IPTG overnight. Cultures were harvested by centrifugationat 6000×g for 10 min. The pellet was then washed with dH₂O andresuspended in buffer (20% sucrose, 20 mM Tris, pH 8.0, 50 μM/mLlysozyme) and incubated at 25° C. for 10 minutes. The lysate was thensonicated for 2×30 seconds and centrifuged at 15000×g for 20 minutes.The pellet was resuspended in resuspension buffer (4.5 M guanidine-HCl,0.1M NaCl, 20 mM Tris, pH 8.0) and incubated for 1 hour at 25° C. Thesuspension was then centrifuged (20 minutes, 15000×g), and thesupernatant was loaded on a NI-NTA Superflow column (Qiagen)pre-equilibrated with 5 volumes of resuspension buffer. The column waswashed with 5 column volumes of resuspension buffer containing 10 mMimidazole. The protein was then eluted using resuspension buffercontaining 225 mM imidazole. The eluate was dialysed against 0.1 M NaCl,20 mM Tris, pH 8.0, and 2% LDAO (Fluka) overnight, and then concentratedvia centrifugation to 4 mg/mL.

To assay direct binding of erastin analogs, 40 μg of purified VDAC2 wasresuspended in 100 μL of Binding Buffer (25 mM HEPES, pH 8.0, 0.1% BSA,7 mM MgCl₂, 15 mM NaCl), and incubated in the presence of 20 μMradiolabelled erastin A9 and erastin A9 or erastin A8 for 15 minutes.The mixture was then deposited onto Protran BA85 0.45 μM binding filters(Whatman) using vacuum filtration. The filter was rinsed 5 times with 1mL wash buffer (25 mM HEPES, 0.1% BSA), and then incubated in 5 mLscintillation liquid (Cytoscint, MP Biomedicals). Radioactivity wasdetected on a LKB Wallac 1211 RACKBETA Liquid Scintillation Counter.

Table 2. Activity of RAS-selective lethal compound erastin in tumor celllines. IC₅₀ values (ng/mL) are shown using the Alamar Blue viabilityassay. BJ engineered tumor cells express TERT, LT, ST, and oncogenicRAS. Isogenic RAS^(V12) lacking cells are identical but lack HRAS^(V12).BJ-DRD cells are derived from BJ cells and contain TERT, oncogenic RAS,a truncated from of p53 (p53DD) that disrupts the tetramerization ofendogenous p53, a CDK4(R24C) mutant resistant to inhibition by p16INK4Aand p151NK4B (the major negative regulators of CDK4) and cyclin D1.These latter protein substitute for LT. HCT-116, A549, Calu-1, and MIAPaCa-2 are tumor cells derived from cancers (colon, lung, lung andpancreatic, respectively) known to have activating mutations in RAS.

Cell Line erastin BJ-TERT/LT/ST/RAS^(V12) 1250 BJ-TERT/LT/ST >20,000 RASselectivity >16 BJ-TERT/LT/RAS^(V12)/ST 1250 BJ-DRD (+RAS) 2500 HCT-116(+KRAS) >20,000 A549 (+KRAS) >20,000 Calu-1 (+KRAS) 1000 MIA PaCa-2(+KRAS) 5000

TABLE 3 Antimycin and 2-ME partially suppress erastin-induced death.Treatment Cell death (%) SD (%) DMSO 0 6 Erastin A1 100 0 Antimycin 51 52-ME 66 3 erastin + antimycin 59 5 erastin + 2-ME 76 5BJ-TERT/LT/ST/RAS^(V12) cells were treated with 0.1% DMSO, 13 μM erastinA1, 23 μM antimycin, 126 μM 2-methoxyestradiol (2-ME) or thecombinations listed and viable cells counted using a hemacytometer.

TABLE 4 Response of tumor cell lines to erastin. Max % EC50 Cell linekilling (uM) Tumor type HOS 100 17 Osteosarcoma SJSA-1 100 12Osteosarcoma BJELR 100 6 foreskin fibroblasts w/TERT, LT, ST, RASSK-LMS-1 100 6 Leimyosarcoma, vulva MES-SA 100 3 Uterine sarcoma HT108098 2 Fibrosarcoma SK-ES-1 96 7 Ewing sarcoma U-2 OS 96 6 OsteosarcomaSK-N-MC 95 10 Neuroepithelioma HeLa 94 0.6 Cervical carcinoma TC71 92 10Ewing sarcoma Hs51.T 88 12 Spindle cell sarcoma TC32 88 8 Peripheralneuroepithelioma Hs 925.T 83 17 Pagetoid sarcoma U973 73 10 Acutemyelogenous leukemia SK-UT 73 4 Uterine, mixed mesodermal tumor MX2 7118 Uterine Sarcoma A673 54 30 Rhabdomyosarcoma EWS502 42 10 Ewingsarcoma LNCaP 32 6 prostate carcinoma BJEH 22 10 foreskin fibroblastw/TERT C-33A 21 0.6 cervical carcinoma SVR 20 2.5 Pancreatic carcinomaA549 0 — lung carcinoma HCT 116 0 — colorectal carcinoma HL-60 0 — acutepromyelocytic leukemia SW982 0 — synovial sarcoma SW872 0 — LiposarcomaA431NS 0 — Epidermoid carcinoma

TABLE 5 Correlation between erastin sensitivity and phospho-ERK level.Cell line Erastin Sensitivity Phospho ERK1/2 A673 0.54 0.66 BJ-TERT 0.220.09 BJ-TERT/LT/ 1.00 0.92 ST/RAS^(V12) EWS 502 0.42 0.12 HL 60 0.000.10 HT 1080 0.98 0.53 SKES1 0.96 0.25 SK N MC 0.95 0.05 SW 872 0.000.12 TC 32 0.88 0.12 TC 71 0.92 0.23 U937 0.73 0.27 The maximum percentkilling induced by erastin in each cell line is shown, along with thelevel of phosph-ERK1/2. The correlation is 0.41.

TABLE 6 Primer sequences Gene Primer Forward primer Reverse primer VDAC1VDAC1 5′-CCTGGACAGCAGGAA 5′-AGGCGTCAGGGTCAA ACAGTAAC-3′ TCTGA-3′ (SEQ IDNO:30) (SEQ ID NO:35) VDAC2 VDAC2 5′-TGATTTTGCTGGACC 5′-CAGCAAGCCAGCCCTGCAA-3′ TCAT-3′ (SEQ ID NO:31) (SEQ ID NO:36) VDAC3 VDAC35′-AATTTCGCCCTGGGT 5′-TCAGTGCCATCGTT TACAA-3′ CACATGT-3′ (SEQ ID NO:32)(SEQ ID NO:37) RPLPO RPLPO.1 5′-ACGGGTACAAACGAG 5′-GCCTTGACCTTTTCTCCTG-3′ AGCAAG-3′ (SEQ ID NO:33) (SEQ ID NO:38) RPLPO RPLPO.25′-GCGACCTGGAAGTCC 5′-ATCTGCTGCATCTG AACTA-3′ CTTGG-3′ (SEQ ID NO:34)(SEQ ID NO:39)

TABLE 7 Sequences of shRNAs Gene Name of sh RNA SEQUENCE VDAC1 V1.161CCGGGCTATGGATTTGGCTTAATAACTCG AGTTATTAAGCCAAATCCATAGCTTTTT (SEQ IDNO: 1) V1.279 CCGGCAAGTACAGATGGACTGAGTACTCG AGTACTCAGTCCATCTGTACTTGTTTTT(SEQ ID NO: 2) V1.396 CCGGCGATTCATCCTTCTCACCTAACTCGAGTTAGGTGAGAAGGATGAATCGTTTTT (SEQ ID NO: 3) V1.607CCGGGCAGTTGGCTACAAGACTGATCTCG AGATCAGTCTTGTAGCCAACTGCTTTTT (SEQ ID NO:4) V1.921 CCGGGCTTGGTCTAGGACTGGAATTCTCG AGAATTCCAGTCCTAGACCAAGCTTTTT(SEQ ID NO: 5) VDAC2 V2.A8(A7) CCGGGCAGCTAAATATCAGTTGGATCTCGAGATCCAACTGATATTTAGCTGCTTTTTG (SEQ ID NO: 6) V2.A9CCGGCAAGGTTTGAAACTGACATTTCTCG AGAAATGTCAGTTTCAAACCTTGTTTTTG (SEQ ID NO:7) V2.A10 CCGGCACTGCTTCCATTTCTGCAAACTCG AGTTTGCAGAAATGGAAGCAGTGTTTTTG(SEQ ID NO: 8) V2.A11 CCGGGTGTGAGTATGGTCTGACTTTCTCGAGAAAGTCAGACCATACTCACACTTTTTG (SEQ ID NO: 9) V2.A12CCGGGTCAACAACTCTAGCTTAATTCTCG AGAATTAAGCTAGAGTTGTTGACTTTTTG (SEQ ID NO:10) VDAC3 V3.B1 CCGGGCAACCTAGAAACCAAATATACTCGAGTATATTTGGTTTCTAGGTTGCTTTTTG (SEQ ID NO: 11) V3.B2CCGGCCAGGAGTCAAATTGACTTTACTCG AGTAAAGTCAATTTGACTCCTGGTTTTTG (SEQ ID NO:12) V3.B3 CCGGCCAAACTGTCACAGAATAATTCTCG AGAATTATTCTGTGACAGTTTGGTTTTTG(SEQ ID NO: 13) V3.B4 CCGGCCAGAATTGGAACACAGACAACTCGAGTTGTCTGTGTTCCATTTCTGGTTTTTG (SEQ ID NO: 14) V3.B5CCGGCAGGAGTCAAATTGACTTTATCTCG AGATAAAGTCAATTTGACTCCTGTTTTTG (SEQ ID NO:15) V3.B6 CCGGCCAGAAGGTGAATGAGAAGATCTCG AGATCTTCTCATTCACCTTCTGGTTTTG(SEQ ID NO: 16) NRAS Nras.304 CCGGCGCACTGACAATCCAGCTAATCTCGAGATTAGCTGGATTGTCAGTGCGTTTTTG (SEQ ID NO: 17) Nras.398CCGGGAAACCTGTTTGTTGGACATACTCG AGTATGTCCAACAAACAGGTTTCTTTTTG (SEQ ID NO:18) Nras.445 CCGGCAGTGCCATGAGAGACCAATACTCG AGTATTGGTCTCTCATGGCACTGTTTTTG(SEQ ID NO: 19) Nras.501 CCGGCCATCAATAATAGCAAGTCATCTCGAGATGACTTGCTATTATTGATGGTTTTTG (SEQ ID NO: 20) Nras.655CCGGCAAGAGTTACGGGATTCCATTCTCG AGAATGGAATCCCGTAACTCTTGTTTTTG (SEQ ID NO:21) KRAS Kras.269 CCGGGACGAATATGATCCAACAATACTCGAGTATTGTTGGATCATATTCGTCTTTTTG (SEQ ID NO: 22) Kras.407CCGGGAGGGCTTTCTTTGTGTATTTCTCG AGAAATACACAAAGAAAGCCCTCTTTTTG (SEQ ID NO:23) Kras.509 CCGGCCTATGGTCCTAGTAGGAAATCTCG AGATTTCCTACTAGGACCATAGGTTTTTG(SEQ ID NO: 24) Kras.667 CCGGGATCCGACAATACAGATTGAACTCGAGTTCAATCTGTATTGTCGGATCTTTTTG (SEQ ID NO: 25) Kras.1160CCGGTAGTTGGAGCTGGTGGCGTAGCTCG AGCTACGCCACCAGCTCCAACTATTTTTG (SEQ ID NO:26) BRAF iBRAF-1 CCGGGAGTTCAGGAGAGTAGCAATTCAAGAGATTGCTACTCTCCTGAACTCTTTTTG (SEQ ID NO: 27) IBRAF exon 5

Synthesis of Erastin Analogs

With reference to FIG. 47, anthranilic acid (28) (40 g, 0.291 mol) wasplaced in a 500 mL round-bottom flask fitted with an addition funnel anda magnetic stir bar. 250 mL of DMF were added to the anthranilic acidand the reaction mixture was placed in an ice bath. Propionyl chloride(1.1 eq., 28 mL, 0.320 mol) was placed in the addition funnel and slowlyadded to the reaction mixture under nitrogen atmosphere. Then thereaction mixture was stirred for 2 h at room temperature. DMF wasremoved under vacuum and an oily residue was suspended in water. Theprecipitated product was collected by filtration, washed with cold waterand dried under vacuum, yielding N-propionyl anthranilic acid (29) as awhite powder (yield: 44.693 g, 79.4%).

N-propionyl anthranilic acid (29) (38.4 g, 0.199 mol) was dissolved inacetic anhydride (150 mL) in a 250 mL round-bottom flask equipped with amagnetic stir bar and a Claisen-distillation head. The flask was heatedat 170-180° C. for 3 h and acetic acid forming in the reaction wasdistilled off. The completion of the reaction was confirmed by LC-MS andTLC analyses. The reaction mixture was cooled to room temperature andacetic anhydride was removed under vacuum. An oily residue wastriturated with hexane. The precipitated product was collected byfiltration, yielding 2-ethyl-benzo[d][1,3]oxazin-4-one (30) as a whitepowder (yield: 29.614 g, 84.9%).

2-Ethyl-benzo[d][1,3]oxazin-4-one (30) (29.614 g, 0.169 mol) was placedin a 250 mL round-bottom flask with a reflux condenser. 100 mL ofchloroform and o-phenetidine (1.1 eq., 25.500 g, 0.186 moles) were addedto the reaction mixture. The reaction mixture was refluxed for 24 h.Then chloroform was removed under vacuum. Ethylene glycol (50 mL) andNaOH (300 mg) were added to the off-white residue and the flask wasequipped with a Cailsen-distillation apparatus. The reaction mixture washeated at 130-140° C. for 5 h and water forming in the reaction wasremoved by distillation. Then the reaction mixture was cooled to roomtemperature and left standing overnight. The precipitated product wasfiltered off, rinsed with cold water and recrystallized from isopropanolto provide 3-(2-ethoxy-phenyl)-2-ethyl-3H-quinazolin-4-one (31) (yield:39.450 g, 79.3%).

3-(2-ethoxy-phenyl)-2-ethyl-3H-quinazolin-4-one (31) (500 mg, 1.699mmol) and sodium acetate (769 mg) were dissolved in 10 mL of glacialacetic acid in a 50 mL round-bottom flask equipped with a magnetic stirbar and an addition funnel. Br₂ (1 eq., 88 μl, 1.699 mmol) was dissolvedin 5 mL of glacial acetic acid and placed in an addition funnel. Thereaction mixture was warmed up to 40° C. and Br₂ solution was addeddropwise to the reaction mixture for 1 h. LC-MS analysis indicatedincomplete reaction. Therefore, another 10 μl of Br₂ were added to thereaction and the reaction mixture was heated for 1 h at 40° C. Aftercompletion of the reaction, acetic acid was removed under vacuum; theresidue was dissolved in 20 mL of chloroform and extracted with water(3×10 mL). The organic phase was separated, dried over anhydrous Na₂SO₄,and filtered through a short silica plug. Chloroform was removed undervacuum to provide2-(1-bromo-ethyl)-3-(2-ethoxy-phenyl)-3H-quinazolin-4-one (32) (yield:561 mg, 88.5%).

2-(1-bromo-ethyl)-3-(2-ethoxy-phenyl)-3H-quinazolin-4-one (32) (500 mg,1.339 mmol) and piperazine (3 eq., 346 mg, 4.016 mmol) were dissolved in30 mL of ethanol in a 50 mL round-bottom flask equipped with a refluxcondenser. The reaction mixture was refluxed for 24 h. Ethanol wasremoved under vacuum; the crude product was dissolved in 30 mL ofchloroform and extracted with water (3×10 mL). The organic phase wasseparated, chloroform was removed under vacuum and the crude product waspurified via rotary chromatography to provide3-(2-ethoxy-phenyl)-2-(1-piperazin-1-yl-ethyl)-3H-quinazolin-4-one (33)(yield: 218 mg, 43.0%).

3-(2-ethoxy-phenyl)-2-(1-piperazin-1-yl-ethyl)-3H-quinazolin-4-one (33)(200 mg, 0.528 mmol) and acetic anhydride (1.2 eq., 65 μm, 0.634 mmol)were dissolved in 5 mL of chloroform and stirred at room temperature for30 min. LC-MS analysis indicated a complete reaction. The crude reactionmixture was extracted with 5% Na₂CO₃ (2-3 mL) and water (2×3 mL). Theorganic phase was separated, dried over anhydrous Na₂SO₄. Na₂SO₄ wasfiltered off and chloroform was removed under vacuum to provide2-[1-(4-Acetyl-piperazin-1-yl)-ethyl]-3-(2-ethoxy-phenyl)-3H-quinazolin-4-one(34) in a quantitative yield.

3-(2-Ethoxy-phenyl)-7-nitro-2-(1-piperazin-1-yl-ethyl)-3H-quinazolin-4-one(51) was synthesized using same methodology as3-(2-ethoxy-phenyl)-2-(1-piperazin-1-yl-ethyl)-3H-quinazolin-4-one (33),except, 4-nitroanthranilic acid (2-amino-4-nitrobenzoic acid) (92a) wasused as a starting material.

3-(2-Ethoxy-phenyl)-6-nitro-2-(1-piperazin-1-yl-ethyl)-3H-quinazolin-4-one(52) was synthesized using same methodology as3-(2-ethoxy-phenyl)-2-(1-piperazin-1-yl-ethyl)-3H-quinazolin-4-one (33),except, 5-nitroanthranilic acid (2-amino-5-nitrobenzoic acid) (92b) wasused as a starting material.

3-(2-Ethoxy-phenyl)-7-nitro-2-(1-piperazin-1-yl-ethyl)-3H-quinazolin-4-one(51) (799 mg, 1.889 mmol), 4-chlorophenoxyacetic acid (1.1 eq., 388 mg,2.078 mmol) and TBTU (1.1 eq., 667 mg, 2.078 mmol) were dissolved in 10mL of chloroform. 200 μl of DIPEA were added to the reaction mixture andit was stirred at room temperature for 20 min. LC-MS and TLC analysesconfirmed completion of the reaction. Chloroform and DIPEA were removedunder vacuum; the crude product was purified via column chromatographyto provide2-(1-{4-[2-(4-chloro-phenoxy)-acetyl]-piperazin-1-yl}-ethyl)-3-(2-ethoxy-phenyl)-7-nitro-3H-quinazolin-4-one(2) (nitro-erastin) in a quantitative yield.

2-(1-{4-[2-(4-chloro-phenoxy)-acetyl]-piperazin-1-yl}-ethyl)-3-(2-ethoxy-phenyl)-7-nitro-3H-quinazolin-4-one(2) (300 mg, 0.507 mmol) was dissolved in 15 mL of methanol in a 25 mLround-bottom flask equipped with a reflux condenser. 300 mg of NH₄HCO₂and 30 mg of Pd/C were added to the reaction mixture and it was refluxedfor 12 h under nitrogen atmosphere. LC-MS and TLC analyses confirmed acomplete reaction. Pd/C was filtered off, methanol was removed undervacuum. The residue was dissolved in 20 mL of chloroform and extractedwith water (3×10 mL), the organic phase was separated and chloroform wasremoved under vacuum. The crude product was purified via a rotarychromatography to provide7-amino-2-(1-{4-[2-(4-chloro-phenoxy)-acetyl]-piperazin-1-yl}-ethyl)-3-(2-ethoxy-phenyl)-3H-quinazolin-4-one(1) (yield: 269 mg, 94.4%).

7-amino-2-(1-{4-[2-(4-chloro-phenoxy)-acetyl]-piperazin-1-yl}-ethyl)-3-(2-ethoxy-phenyl)-3H-quinazolin-4-one(1) (25 mg, 0.0445 mmol) was dissolved in 2 mL of chloroform in a 6 mLvial. Acetic anhydride (5 eq., 22 μl, 0.222 mmol) was added to thereaction mixture and it was heated at 50° C. for 30 min. LC-MS and TLCanalyses confirmed a complete reaction. Chloroform and acetic anhydridewere removed under vacuum. The residue was dissolved in 3 mL ofchloroform and extracted with 5% Na₂CO₃ (1 mL) and water (1 mL). Theorganic phase was separated and dried over anhydrous Na₂SO₄. Na₂SO₄ wasfiltered off and chloroform was removed under vacuum to provideN-[2-(1-{4-[2-(4-chloro-phenoxy)-acetyl]-piperazin-1-yl}-ethyl)-3-(2-ethoxy-phenyl)-4-oxo-3,4-dihydro-quinazolin-7-yl]-acetamide(3) in a quantitative yield.

3-(2-methoxyphenyl)-2-(1-(piperazin-1-yl)ethyl)quinazolin-4(3H)-one (37)was synthesized using same methodology as3-(2-ethoxy-phenyl)-2-(1-piperazin-1-yl-ethyl)-3H-quinazolin-4-one (33),except, 2-methoxyaniline (38) was used instead of o-phenetidine.

3-(2-methoxyphenyl)-2-(1-(piperazin-1-yl)ethyl)quinazolin-4(3H)-one (37)(1 g, 2.77 mmol) was dissolved in 30 mL of DMF and EtSNa (2.5 eq., 576mg, 6.86 mmol) was added to the reaction mixture. The reaction mixturewas heated at 70-80° C. for 2 hr. LC-MS and TLS analysis indicatedincomplete deprotection, therefore, an additional EtSNa (1.25 eq, 288mg, 3.43 mmol) was added to the reaction mixture and it was heated for 3h. The reaction mixture was diluted with 50 mL of CHCl₃ and twiceextracted with 30 mL of H₂O, the organic phase was separated andsolvents were removed under vacuum. The crude product was purified viacolumn chromatography to provide3-(2-hydroxyphenyl)-2-(1-(piperazin-1-yl)ethyl)quinazolin-4(3H)-one(39).

3-(2-hydroxyphenyl)-2-(1-(piperazin-1-yl)ethyl)quinazolin-4(3H)-one (39)(1 eq., 124 mg, 0.353 mmol) was dissolved in 20 mL of CHCl₃, TEA (1.1eq, 0.388 mmol) and 2-(4-chlorophenoxy)acetyl chloride (1.1 eq, 80 mg,0.388 mmol) were added to the reaction mixture. The reaction mixture wasstirred at RT for 1 hr, LC-MS analysis indicated complete consumption ofthe starting material. The reaction mixture was diluted with 20 mL ofCHCl₃ and extracted with H₂O (3×10 mL). The organic phase was separated,dried over Na₂SO₄ and solvent was removed under vacuum. The crudeproduct was purified via column chromatography to provide2-(1-(4-(2-(4-chlorophenoxy)acetyl)piperazin-1-yl)ethyl)-3-(2-hydroxyphenyl)quinazolin-4(3H)-one(40) (110 mg, 60% yield).

Characterization:

Compounds are mixtures of diastereomers

Erastin (Compound 36): ¹H-NMR (300 MHz, CDCl₃):

8.30-8.27 p.p.m. (m, 1H), 7.80-7.72 (m, 2H), 7.50-7.40 (m, 2H),7.40-7.00 (m, 5H), 6.84 (d, J=9.0 Hz, 2H), 4.61 (s, 2H), 4.05-3.98 (m,2H), 3.55-3.30 (m, 5H), 2.80-2.40 (m, 2H), 2.25-1.95 (m, 2H), 1.31 (t,J=6.9 Hz, 3H), 1.23-1.17 (m, 3H); ¹³C-NMR (75 MHz, CDCl₃): ō 165.87,161.80, 156.53, 156.41, 155.79, 154.77, 153.65, 146.98, 134.21, 131.51,130.52, 129.43, 128.59, 127.57, 126.99, 126.79, 126.74, 126.55, 126.21,121.38, 121.09, 120.76, 120.38, 115.92, 112.99, 112.67, 67.80, 67.74,64.15, 64.08, 60.25, 59.83, 48.85, 48.50, 48.32, 45.54, 45.29, 42.02,14.75, 14.58, 12.95, 10.61; HRMS (m/z): [M]⁺ calcd for C₃₀H₃₂ClN₄O₄,547.2112; found, 547.2083.

Erastin A6 (Compound 57): ¹H-NMR (300 MHz, CDCl₃):

8.28 p.p.m. (d, J=7.8, 1H), 7.69-7.66 (m, 2H), 7.46-7.23 (m, 2H), 7.26(s, 1H), 7.22 (d, J=8.4 Hz, 2H), 7.19-7.00 (m, 3H), 6.87 (d, J=8.7 Hz,2H), 4.61 (s, 2H), 4.03 (q, J=6.9 Hz, 2H), 3.81 (s, 2H), 3.59 (q, J=6.9Hz, 1H), 3.51-3.30 (m, 4H), 2.52-2.49 (m, 2H), 2.25-2.00 (m, 2H), 1.88(s, 6H), 1.31 (t, J=6.6 Hz), 1.28-1.15 (m, 3H); ¹³C-NMR (75 MHz, CDCl₃):ō 166.25, 161.83, 156.85, 156.56, 154.82, 147.01, 135.62, 134.19,131.56, 130.52, 128.59, 128.56, 127.60, 127.01, 126.79, 126.74, 126.25,121.11, 120.73, 120.40, 114.66, 112.99, 112.67, 67.65, 64.16, 64.08,60.29, 59.84, 48.95, 48.80, 48.42, 45.58, 45.26, 41.99, 14.78, 14.59,12.86, 10.63; HRMS (m/z): [M]⁺ calcd for C₃₁H₃₆N₅O₄, 542.2767; found,542.2751.

Erastin A8 (Compound 34): ¹H-NMR (300 MHz, CDCl₃):

8.29 p.p.m. (d, J=8.1 Hz, 1H), 7.85-7.65 (m, 2H), 7.51-7.30 (m, 3H),7.33 (dd, J=7.8 Hz, J=1.9 Hz, 1H), 7.15 (dd, J=7.9 Hz, J=1.5 Hz, 1H),7.11-6.99 (m, 3H), 4.10-3.95 (m, 2H), 3.60-3.15 (m, 5H), 2.65-2.45 (m,2H), 2.25-2.10 (m, 2H), 2.03 (s, 3H), 1.33 (t, J=6.6 Hz, 3H), 1.25-1.10(m, 3H); ¹³C-NMR (75 MHz, CDCl₃): ō 174.27, 169.43, 169.36, 163.28,162.27, 157.11, 156.35, 155.18, 154.06, 147.43, 134.63, 131.97, 130.95,129.00, 127.99, 127.40, 127.20, 127.15, 126.58, 125.92, 121.78, 121.49,121.14, 120.81, 113.38, 113.11, 64.56, 64.50, 60.74, 60.27, 49.30,48.91, 48.71, 46.93, 46.64, 42.06, 41.75, 21.57, 21.23, 15.15, 14.99,13.50, 11.16; HRMS (m/z): [M]⁺ calcd for C₂₄H₂₉N₄O₃, 421.2240; found,421.2247.

Erastin A9 (Compound 3): ¹H-NMR (300 MHz, CDCl₃): ō 8.19 p.p.m. (d,J=8.7 Hz, 1H), 8.12 (s, 1H), 8.06 (s, 1H); 7.51 (d, J=8.7 Hz, 1H), 7.43(t, J=5.2 Hz, 1H), 7.28 (s, 1H), 7.26-7.18 (m, 3H), 7.15 (dd, J=7.7 Hz,J=1.8 Hz, 1H), 7.07 (d, J=7.5 Hz, 1H), 7.02 (d, J=7.5 Hz, 1H), 6.89-6.80(m, 2H), 4.64 (s, 2H), 4.01 (q, J=6.9 Hz, 2H), 3.59 (q, J=6.6 Hz, 1H),3.50-3.30 (m, 4H), 2.70-2.60 (m, 1H), 2.55-2.45 (m, 1H), 2.30-2.20 (m,2H), 2.19 (s, 3H), 1.31 (d, J=6.6 Hz, 3H), 1.19 (t, J=6.9 Hz, 3H);¹³C-NMR (75 MHz, CDCl₃): δ 169.54, 166.43, 161.93, 157.40, 156.79,155.16, 148.62, 144.16, 131.02, 129.85, 129.05, 128.29, 126.99, 126.53,121.20, 119.23, 117.09, 116.82, 116.35, 113.36, 68.02, 64.54, 60.53,49.11, 45.72, 42.55, 24.96, 15.13, 13.51; HRMS (m/z): [M]⁺ calcd forC₃₂H₃₅ClN₅O₅, 604.2327; found, 604.2356.

Erastin B2 (Compound 54): ¹H-NMR (300 MHz, CD₃SOCD₃): δ 7.32-7.25 p.p.m.(m, 4H), 7.13 (d, J=7.8 Hz, 2H), 6.93 (d, J=9.0 Hz, 2H), 4.82 (s, 2H),4.41 (s, 2H), 3.90-3.70 (m, 1H), 3.68 (s, 2H), 3.50-3.30 (m, 6H),2.50-2.34 (m, 6H), 1.07 (d, J=6.6 Hz, 6H); ¹³C-NMR (75 MHz, CD₃SOCD₃): δ166.38, 158.10, 157.82, 143.57, 137.90, 129.93, 127.97, 127.86, 125.34,117.26, 66.85, 57.38, 53.88, 53.56, 46.18, 44.91, 44.05, 42.72, 42.09,23.96; HRMS (m/z): [M]⁺ calcd for C₂₆H₃₇ClN₅O₃, 502.2585; found,502.2603.

In the present invention, the following nomenclature is followed:Erastin (36) is2-(1-{4-[2-(4-Chloro-phenoxy)-acetyl]-piperazin-1-yl}-ethyl)-3-(2-ethoxy-phenyl)-3H-quinazolin-4-one.Erastin A6 (57) is2-(1-{4-[2-(4-Aminomethyl-phenoxy)-acetyl]-piperazin-1-yl}-ethyl)-3-(2-ethoxy-phenyl)-3H-quinazolin-4-one.Erastin A8 (34) isN-[2-(1-{4-[2-(4-Chloro-phenoxy)-acetyl]-piperazin-1-yl}-ethyl)-3-(2-ethoxy-phenyl)-4-oxo-3,4-dihydro-quinazolin-7-yl]-acetamide.Erastin A9 (3) is2-[1-(4-Acetyl-piperazin-1-yl)-ethyl]-3-(2-ethoxy-phenyl)-3H-quinazolin-4-one.Erastin B2 (54) is1-(4-Aminomethyl-benzyl)-1-(2-{4-[2-(4-chloro-phenoxy)-acetyl]-piperazin-1-yl}-ethyl)-3-isopropyl-urea.

CITED LITERATURE

All documents cited, herein, including those cited below areincorporated by reference as if recited in full herein.

-   1. Shawver, L. K., Slamon, D. & Ullrich, A. Smart drugs: tyrosine    kinase inhibitors in cancer therapy. Cancer Cell. 1: 117-23. (2002).-   2. Capdeville, R., Buchdunger, E., Zimmermann, J. & Matter, A.    Glivec (ST1571, imatinib), a rationally developed, targeted    anticancer drug. Nat Rev Drug Discov. 1: 493-502. (2002).-   3. Mokbel, K. & Hassanally, D. From HER2 to herceptin. Curr Med Res    Opin. 17: 51-9. (2001).-   4. Downward, J. Targeting RAS signalling pathways in cancer therapy.    Nat Rev Cancer. 3: 11-22 (2003).-   5. Kaelin, W. G., Jr. The concept of synthetic lethality in the    context of anticancer therapy. Nat Rev Cancer. 5: 689-98 (2005).-   6. Shi, Y. et al. Enhanced sensitivity of multiple myeloma cells    containing PTEN mutations to CCI-779. Cancer Res. 62: 5027-34.    (2002).-   7. Druker, B. J. et al. Effects of a selective inhibitor of the Abl    tyrosine kinase on the growth of Bcr-Abl positive cells. Nat. Med.    2: 561-6. (1996).-   8. Dolma, S., Lessnick, S. L., Hahn, W. C. & Stockwell, B. R.    Identification of genotype-selective antitumor agents using    synthetic lethal chemical screening in engineered human tumor cells.    Cancer Cell. 3: 285-96 (2003).-   9. Stockwell, B. R., Haggarty, S. J. & Schreiber, S. L.    High-throughput screening of small molecules in miniaturized    mammalian cell-based assays involving post-translational    modifications. Chem. Biol. 6: 71-83. (1999).-   10. Bailey, S, N., Sabatini, D. M. & Stockwell, B. R. Microarrays of    Small Molecules Embedded in Biodegradable Polymers for Use in    Mammalian Cell-Based Screens. Proc Nat Acad Sci USA. 101:    16144-16149 (2004).-   11. Torrance, C. J., Agrawal, V., Vogelstein, B. & Kinzler, K. W.    Use of isogenic human cancer cells for high-throughput screening and    drug discovery. Nat Biotechnol. 19: 940-5. (2001).-   12. Colicelli, J. Human RAS superfamily proteins and related    GTPases. Sci STKE. 2004: RE13 (2004).-   13. Walker, K. & Olson, M. F. Targeting Ras and Rho GTPases as    opportunities for cancer therapeutics. Curr Opin Genet Dev. 15: 62-8    (2005).-   14. Schreiber, S. L. Chemical genetics resulting from a passion for    synthetic organic chemistry. Bioorg. Med. Chem. 6:1127-1152 (1998).-   15. Schreiber, S. L. Target-oriented and diversity-oriented organic    synthesis in drug discovery. Science. 287: 1964-9. (2000).-   16. Schreiber, S. L. The small-molecule approach to biology:    Chemical genetics and diversity-oriented organic synthesis make    possible the systematic exploration of biology. Chem. & Eng. News.    81: 51-61 (2003).-   17. Stockwell, B. R. Chemical genetics: ligand-based discovery of    gene function. Nat Rev Genet. 1: 116-25. (2000).-   18. Stockwell, B. R. Frontiers in chemical genetics. Trends    Biotechnol. 18: 449-55. (2000).-   19. Stockwell, B. R. Chemical genetic screening approaches to    neurobiology. Neuron. 36: 559-62 (2002).-   20. Stockwell, B. R. Exploring biology with small organic molecules.    Nature. 432: 846-54 (2004).-   21. Smukste, I. & Stockwell, B. R. Advances in chemical genetics.    Annu Rev Genomics Hum Genet. 6: 261-86 (2005).-   22. Brown, E. J., et al. & Schreiber, S. L. A mammalian protein    targeted by G1-arresting rapamycin-receptor complex. Nature. 369:    756-758 (1994).-   23. Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P. &    Snyder, S. H. RAFT1: a mammalian protein that binds to FKBP12 in a    rapamycin-dependent fashion and is homologous to yeast TORs. Cell.    78: 35-43. (1994).-   24. Khersonsky, S. M. et al. Facilitated forward chemical genetics    using a tagged triazine library and zebrafish embryo screening. J Am    Chem Soc. 125: 11804-5 (2003).-   25. Williams, D. et al. Identification of compounds that bind    mitochondrial F1F0 ATPase by screening a triazine library for    correction of albinism. Chem Biol. 11: 1251-9 (2004).-   26. Wan, Y. et al. Synthesis and target identification of    hymenialdisine analogs. Chem Biol. 11: 247-59 (2004).-   27. Oda, Y. et al. Quantitative chemical proteomics for identifying    candidate drug targets. Anal Chem. 75: 2159-65 (2003).-   28. Parsons, A. B. et al. Integration of chemical-genetic and    genetic interaction data links bioactive compounds to cellular    target pathways. Nat Biotechnol. 22: 62-69 (2003).-   29. Hsiang, Y. H. & Liu, L. F. Identification of mammalian DNA    topoisomerase I as an intracellular target of the anticancer drug    camptothecin. Cancer Res. 48: 1722-6. (1988).-   30. Eng, W. K., Faucette, L., Johnson, R. K. & Sternglanz, R.    Evidence that DNA topoisomerase I is necessary for the cytotoxic    effects of camptothecin. Mol Pharmacol. 34: 755-60. (1988).-   31. Madden, K. R. & Champoux, J. J. Overexpression of human    topoisomerase I in baby hamster kidney cells: hypersensitivity of    clonal isolates to camptothecin. Cancer Res. 52: 525-32. (1992).-   32. Andoh, T. et al. Characterization of a mammalian mutant with a    camptothecin-resistant DNA topoisomerase I. Proc Natl Acad Sci USA.    84: 5565-9. (1987).-   33. Bjornsti, M. A., Benedetti, P., Viglianti, G. A. & Wang, J. C.    Expression of human DNA topoisomerase I in yeast cells lacking yeast    DNA topoisomerase I: restoration of sensitivity of the cells to the    antitumor drug camptothecin. Cancer Res. 49: 6318-23. (1989).-   34. Champoux, J. J. Structure-based analysis of the effects of    camptothecin on the activities of human topoisomerase I. Ann N Y    Acad Sci. 922: 56-64. (2000).-   35. Liu, L. F. et al. Mechanism of action of camptothecin. Ann N Y    Acad Sci. 922: 1-10. (2000).-   36. D'Arpa, P., Beardmore, C. & Liu, L. F. Involvement of nucleic    acid synthesis in cell killing mechanisms of topoisomerase poisons.    Cancer Res. 50: 6919-24. (1990).-   37. Hsiang, Y. H., Lihou, M. G. & Liu, L. F. Arrest of replication    forks by drug-stabilized topoisomerase I-DNA cleavable complexes as    a mechanism of cell killing by camptothecin. Cancer Res. 49:    5077-82. (1989).-   38. Tsao, Y. P., Russo, A., Nyamuswa, G., Silber, R. & Liu, L. F.    Interaction between replication forks and topoisomerase I-DNA    cleavable complexes: studies in a cell-free SV40 DNA replication    system. Cancer Res. 53: 5908-14. (1993).-   39. Traganos, F., Seiter, K., Feldman, E., Halicka, H. D. &    Darzynkiewicz, Z. Induction of apoptosis by camptothecin and    topotecan. Ann N Y Acad Sci. 803: 101-10. (1996).-   40. Prestwich, G. D., Dorman, G., Elliott, J. T., Marecak, D. M. &    Chaudhary, A. Benzophenone photoprobes for phosphoinositides,    peptides and drugs. Photochem Photobiol. 65: 222-34 (1997).-   41. Olszewski, J. D. et al. Tethered benzophenone reagents for the    synthesis of photoactivatable ligands. Bioconjug Chem. 6: 395-400    (1995).-   42. Dorman, G. & Prestwich, G. D. Benzophenone photophores in    biochemistry. Biochemistry. 33: 5661-73 (1994).-   43. Chen, J., Zheng, X. F., Brown, E. J. & Schreiber, S. L.    Identification of an 11-kDa FKBP12-rapamycin-binding domain within    the 289-kDa FKBP12-rapamycin-associated protein and characterization    of a critical serine residue. Proc Natl Acad Sci USA. 92: 4947-51    (1995).-   44. Stan, R. et al. Interaction between FKBP12-rapamycin and TOR    involves a conserved serine residue. J. Biol. Chem. 269: 32027-32030    (1994).-   45. Harvey, J. J. An Unidentified Virus Which Causes The Rapid    Production Of Tumours In Mice. Nature. 204:1104-5 (1964).-   46. Kirsten, W. H. & Mayer, L. A. Morphological responses to a    murine erythroblastosis virus. J. Natl. Cancer Inst. 39: 311-335    (1967).-   47. Barbacid, M. ras genes. Annu Rev Biochem. 56: 779-827 (1987).-   48. Bos, J. L. ras oncogenes in human cancer: a review. Cancer Res.    49: 4682-9 (1989).-   49. Guerra, C. et al. Tumor induction by an endogenous K-ras    oncogene is highly dependent on cellular context. Cancer Cell. 4:    111-20 (2003).-   50. Jackson, E. L. et al. Analysis of lung tumor initiation and    progression using conditional expression of oncogenic K-ras. Genes    Dev. 15: 3243-8 (2001).-   51. Johnson, L. et al. Somatic activation of the K-ras oncogene    causes early onset lung cancer in mice. Nature. 410: 1111-6 (2001).-   52. Meuwissen, R., Linn, S. C., van der Valk, M., Mooi, W. J. &    Berns, A. Mouse model for lung tumorigenesis through Cre/lox    controlled sporadic activation of the K-Ras oncogene. Oncogene. 20:    6551-8 (2001).-   53. Malumbres, M. & Barbacid, M. RAS oncogenes: the first 30 years.    Nat Rev Cancer. 3: 459-65 (2003).-   54. Coleman, M. L., Marshall, C. J. & Olson, M. F. RAS and RHO    GTPases in G1-phase cell-cycle regulation. Nat Rev Mol Cell Biol. 5:    355-66 (2004).-   55. Hahn, W. C. Immortalization and transformation of human cells.    Mol. Cells. 13: 351-61. (2002).-   56. Hahn, W. C. et al. Creation of human tumour cells with defined    genetic elements. Nature. 400: 464-8. (1999).-   57. Hahn, W. C. et al. Enumeration of the simian virus 40 early    region elements necessary for human cell transformation. Mol Cell    Biol. 22: 2111-23. (2002).-   58. Hahn, W. C. & Weinberg, R. A. Modelling the molecular circuitry    of cancer. Nat Rev Cancer. 2: 331-41. (2002).-   59. Hahn, W. C. & Weinberg, R. A. Rules for making human tumor    cells. N Engl J Med. 347: 1593-603. (2002).-   60. Lessnick, S. L., Dacwag, C. S. & Golub, T. R. The Ewing's    sarcoma oncoprotein EWS/FLI induces a p53-dependent growth arrest in    primary human fibroblasts. Cancer Cell. 1: 393401. (2002).-   61. Rostovtseva, T. K., Tan, W. & Colombini, M. On the Role of VDAC    in Apoptosis: Fact and Fiction. J Bioenerg Biomembr. 37: 129-42    (2005).-   62. Rahmani, Z., Maunoury, C. & Siddiqui, A. Isolation of a novel    human voltage-dependent anion channel gene. Eur J Hum Genet. 6:    337-40 (1998).-   63. Graham, B. H. & Craigen, W. J. Genetic approaches to analyzing    mitochondrial outer membrane permeability. Curr Top Dev Biol. 59:    87-118 (2004).-   64. Anflous, K., Armstrong, D. D. & Craigen, W. J. Altered    mitochondrial sensitivity for ADP and maintenance of    creatine-stimulated respiration in oxidative striated muscles from    VDAC1-deficient mice. J Biol Chem. 276: 1954-60 (2001).-   65. Sampson, M. J. et al. Immotile sperm and infertility in mice    lacking mitochondrial voltage-dependent anion channel type 3. J Biol    Chem. 276: 39206-12 (2001).-   66. Forte, M., Guy, H. R. & Mannella, C. A. Molecular genetics of    the VDAC ion channel: structural model and sequence analysis. J    Bioenerg Biomembr. 19: 341-50 (1987).-   67. Shao, L., Kinnally, K. W. & Mannella, C. A. Circular dichroism    studies of the mitochondrial channel, VDAC, from Neurospora crassa.    Biophys J. 71: 778-86 (1996).-   68. Kmita, H., Budzinska, M. & Stobienia, O. Modulation of the    voltage-dependent anion-selective channel by cytoplasmic proteins    from wild type and the channel depleted cells of Saccharomyces    cerevisiae. Acta Biochim Pol. 50: 415-24 (2003).-   69. Mannella, C. A. Minireview: on the structure and gating    mechanism of the mitochondrial channel, VDAC. J Bioenerg Biomembr.    29: 525-31 (1997).-   70. Thomas, L., Blachly-Dyson, E., Colombini, M. & Forte, M. Mapping    of residues forming the voltage sensor of the voltage-dependent    anion-selective channel. Proc Natl Acad Sci USA. 90: 5446-9 (1993).-   71. Stanley, S., Dias, J. A., D'Arcangelis, D. & Mannella, C. A.    Peptide-specific antibodies as probes of the topography of the    voltage-gated channel in the mitochondrial outer membrane of    Neurospora crassa. J Biol Chem. 270: 16694-700 (1995).-   72. Casadio, R., Jacoboni, I., Messina, A. & De Pinto, V. A 3D model    of the voltage-dependent anion channel (VDAC). FEBS Lett. 520:1-7    (2002).-   73. Chandra, D., Choy, G., Daniel, P. T. & Tang, D. G. Bax-dependent    regulation of Bak by voltage-dependent anion channel 2. J Biol Chem.    280: 19051-61 (2005).-   74. Baker, M. A., Lane, D. J., Ly, J. D., De Pinto, V. & Lawen, A.    VDAC1 is a transplasma membrane NADH-ferricyanide reductase. J Biol    Chem. 279: 4811-9 (2004).-   75. Thinnes, F. P. Evidence for extra-mitochondrial localization of    the VDAC/porin channel in eucaryotic cells. J Bioenerg Biomembr. 24:    71-5 (1992).-   76. Dermietzel, R. et al. Cloning and in situ localization of a    brain-derived porin that constitutes a large-conductance anion    channel in astrocytic plasma membranes. Proc Natl Acad Sci USA. 91:    499-503 (1994).-   77. Thinnes, F. P. et al. Studies on human porin XXI: gadolinium    opens Up cell membrane standing porin channels making way for the    osmolytes chloride or taurine-A putative approach to activate the    alternate chloride channel in cystic fibrosis. Mol Genet Metab. 69:    240-51 (2000).-   78. Buettner, R., Papoutsoglou, G., Scemes, E., Spray, D. C. &    Dermietzel, R. Evidence for secretory pathway localization of a    voltage-dependent anion channel isoform. Proc Natl Acad Sci USA. 97:    3201-6 (2000).-   79. Gonzalez-Gronow, M., Kalfa, T., Johnson, C. E., Gawdi, G. &    Pizzo, S. V. The voltage-dependent anion channel is a receptor for    plasminogen kringle 5 on human endothelial cells. J Biol Chem. 278:    27312-8 (2003).-   80. Bahamonde, M. I., Fernandez-Fernandez, J. M., Guix, F. X.,    Vazquez, E. & Valverde, M. A. Plasma membrane voltage-dependent    anion channel mediates antiestrogen-activated maxi CI-currents in    C1300 neuroblastoma cells. J Biol Chem. 278: 33284-9 (2003).-   81. Bahamonde, M. I. & Valverde, M. A. Voltage-dependent anion    channel localises to the plasma membrane and peripheral but not    perinuclear mitochondria. Pflugers Arch. 446: 309-13 (2003).-   82. Fiek, C., Benz, R., Roos, N. & Brdiczka, D. Evidence for    identity between the hexokinase-binding protein and the    mitochondrial porin in the outer membrane of rat liver mitochondria.    Biochim Biophys Acta. 688: 42940 (1982).-   83. Crompton, M. The mitochondrial permeability transition pore and    its role in cell death. Biochem J. 341 (Pt 2): 23349 (1999).-   84. Brdiczka, D. Contact sites between mitochondrial envelope    membranes. Structure and function in energy- and protein-transfer.    Biochim Biophys Acta. 1071: 291-312 (1991).-   85. Krimmer, T. et al. Biogenesis of porin of the outer    mitochondrial membrane involves an import pathway via receptors and    the general import pore of the TOM complex. J Cell Biol. 152:    289-300 (2001).-   86. Linden, M. & Karlsson, G. Identification of porin as a binding    site for MAP2. Biochem Biophys Res Commun. 218: 833-6 (1996).-   87. Madesh, M. & Hajnoczky, G. VDAC-dependent permeabilization of    the outer mitochondrial membrane by superoxide induces rapid and    massive cytochrome c release. J Cell Biol. 155: 1003-15 (2001).-   88. Tsujimoto, Y. & Shimizu, S. VDAC regulation by the Bcl-2 family    of proteins. Cell Death Differ. 7: 1174-81 (2000).-   89. Shimizu, S., Narita, M. & Tsujimoto, Y. Bcl-2 family proteins    regulate the release of apoptogenic cytochrome c by the    mitochondrial channel VDAC. Nature. 399: 483-7 (1999).-   90. Averet, N., Aguilaniu, H., Bunoust, O., Gustafsson, L. &    Rigoulet, M. NADH is specifically channeled through the    mitochondrial porin channel in Saccharomyces cerevisiae. J Bioenerg    Biomembr. 34: 499-506 (2002).-   91. Vander Heiden, M. G. et al. Outer mitochondrial membrane    permeability can regulate coupled respiration and cell survival.    Proc Natl Acad Sci USA. 97: 4666-71 (2000).-   92. Leist, M. & Jaattela, M. Four deaths and a funeral: from    caspases to alternative mechanisms. Nat Rev Mol Cell Biol. 2: 589-98    (2001).-   93. Majno, G. & Joris, I. Apoptosis, oncosis, and necrosis. An    overview of cell death. Am J Pathol. 146: 3-15. (1995).-   94. Kerr, J. F., Wyllie, A. H. & Currie, A. R. Apoptosis: a basic    biological phenomenon with wide-ranging implications in tissue    kinetics. Br J Cancer. 26: 239-57 (1972).-   95. Baehrecke, E. H. Autophagic programmed cell death in Drosophila.    Cell Death Differ. 10: 940-5 (2003).-   96. Syntichaki, P. & Tavernarakis, N. The biochemistry of neuronal    necrosis: rogue biology? Nat Rev Neurosci. 4: 672-84 (2003).-   97. Jagtap, P. & Szabo, C. Poly(ADP-ribose) polymerase and the    therapeutic effects of its inhibitors. Nat Rev Drug Discov. 4:    421-40 (2005).-   98. Blankenberg, F. G. Recent advances in the imaging of programmed    cell death. Curr Pharm Des. 10: 1457-67 (2004).-   99. Danial, N. N. & Korsmeyer, S. J. Cell death: critical control    points. Cell. 116: 205-19 (2004).-   100. Sgonc, R. & Gruber, J. Apoptosis detection: an overview. Exp    Gerontol. 33: 525-33 (1998).-   101. Walker, N. I., Harmon, B. V., Gobe, G. C. & Kerr, J. F.    Patterns of cell death. Methods Achiev Exp Pathol. 13: 18-54 (1988).-   102. Boyce, M., Degterev, A. & Yuan, J. Caspases: an ancient    cellular sword of Damocles. Cell Death Differ. 11: 29-37 (2004).-   103. Zhivotovsky, B. Apoptosis, necrosis and between. Cell Cycle. 3:    64-6 (2004).-   104. Ohsumi, Y. Molecular dissection of autophagy: two    ubiquitin-like systems. Nat Rev Mol Cell Biol. 2: 211-6. (2001).-   105. Abeliovich, H., Zhang, C., Dunn, W. A., Jr., Shokat, K. M. &    Klionsky, D. J. Chemical genetic analysis of Apg1 reveals a    non-kinase role in the induction of autophagy. Mol Biol Cell. 14:    477-90 (2003).-   106. Broker, L. E., Kruyt, F. A. & Giaccone, G. Cell death    independent of caspases: a review. Clin Cancer Res. 11: 3155-62    (2005).-   107. Chen, Y. et al. Living T9 glioma cells expressing membrane    macrophage colony-stimulating factor produce immediate tumor    destruction by polymorphonuclear leukocytes and macrophages via a    “paraptosis”-induced pathway that promotes systemic immunity against    intracranial T9 gliomas. Blood. 100: 1373-80 (2002).-   108. Fombonne, J., Padron, L., Enjalbert, A., Krantic, S. &    Torriglia, A. A novel paraptosis pathway involving LEI/L-DNasell for    EGF-induced cell death in somato-lactotrope pituitary cells.    Apoptosis. (2006).-   109. Franklin, D. J. & Berges, J. A. Mortality in cultures of the    dinoflagellate Amphidinium carterae during culture senescence and    darkness. Proc Biol Sci. 271: 2099-107 (2004).-   110. Jadus, M. R. et al. Human U251MG glioma cells expressing the    membrane form of macrophage colony-stimulating factor (mM-CSF) are    killed by human monocytes in vitro and are rejected within    immunodeficient mice via paraptosis that is associated with    increased expression of three different heat shock proteins. Cancer    Gene Ther. 10: 411-20 (2003).-   111. Schneider, D. et al. Intracellular acidification by inhibition    of the Na+/H+-exchanger leads to caspase-independent death of    cerebellar granule neurons resembling paraptosis. Cell Death Differ.    11: 760-70 (2004).-   112. Sperandio, S. et al. Paraptosis: mediation by MAP kinases and    inhibition by AIP-1/Alix. Cell Death Differ. 11: 1066-75 (2004).-   113. Wang, Y. et al. An alternative form of paraptosis-like cell    death, triggered by TAJ/TROY and enhanced by PDCD5 overexpression. J    Cell Sci. 117: 1525-32 (2004).-   114. Root, D. E., Flaherty, S. P., Kelley, B. P. & Stockwell, B. R.    Biological mechanism profiling using an annotated compound library.    Chem. Biol. 10: 881-92 (2003).-   115. Blanchard, B. J., Stockwell, B. R. & Ingram, V. M. Eliminating    membrane depolarization caused by the Alzheimer peptide Abeta (1-42,    aggr.). Biochem Biophys Res Commun. 293:1204-8 (2002).-   116. Lunn, M. R. et al. Indoprofen upregulates the survival motor    neuron protein through a cyclooxygenase-independent mechanism.    Chemistry & Biology. 11: 1489-1493 (2004).-   117. Stegmaier, K. et al. Gene expression-based high-throughput    screening (GE-HTS) and application to leukemia differentiation. Nat.    Genet. 36: 257-63 (2004).-   118. Pollitt, S. K. et al. A rapid cellular FRET assay of    polyglutamine aggregation identifies a novel inhibitor. Neuron. 40:    685-94 (2003).-   119. Blanchard, B. J. et al. Efficient reversal of Alzheimer's    disease fibril formation and elimination of neurotoxicity by a small    molecule. Proc Natl Acad Sci USA. 101: 14326-32 (2004).-   120. Root, D. E., Kelley, B. P. & Stockwell, B. R. Detecting spatial    patterns in biological array experiments. J Biomol Screen. 8: 393-8    (2003).-   121. Root, D. E., Kelley, B. P. & Stockwell, B. R. Global analysis    of large-scale chemical and biological experiments. Curr Opin Drug    Discov Devel. 5: 355-60 (2002).-   122. Kelley, B. P. et al. PathBLAST: a tool for alignment of protein    interaction networks. Nucleic Acids Res. 32: W83-8 (2004).-   123. Kelley, B. P. et al. Conserved pathways within bacteria and    yeast as revealed by global protein network alignment. Proc Natl    Acad Sci USA. 100: 11394-9 (2003).-   124. Kelley, B. P. et al. A Flexible Data Analysis Tool for Chemical    Genetic Screens. Chemistry & Biology. 11: 1495-1503 (2004).-   125. Moffat, J. et al. A lentiviral RNAi library for human and mouse    genes applied to an arrayed viral high-content screen. Cell. 124:    1283-98 (2006).-   126. Lunn, M. R. et al. Indoprofen upregulates the survival motor    neuron protein through a cyclooxygenase-independent mechanism. Chem    Biol. 11: 1489-93 (2004).-   127. Kelley, B. P. et al. A flexible data analysis tool for chemical    genetic screens. Chem Biol. 11: 1495-503 (2004).-   128. Smukste, I., Bhalala, O., Persico, M. & Stockwell, B. R. Using    small molecules to overcome drug resistance induced by a viral    oncogene. Cancer Cell. 9: 133-46 (2006).-   129. Dorman, G. & Prestwich, G. D. Using photolabile ligands in drug    discovery and development. Trends Biotechnol. 18: 64-77 (2000).-   130. Weber, P. J. & Beck-Sickinger, A. G. Comparison of the    photochemical behavior of four different photoactivatable probes. J    Pept Res. 49: 375-83 (1997).-   131. Pestic-Dragovich, L. et al. A myosin I isoform in the nucleus.    Science. 290: 337-41 (2000).-   132. Nociari, M. M., Shalev, A., Benias, P. & Russo, C. A novel    one-step, highly sensitive fluorometric assay to evaluate    cell-mediated cytotoxicity. J. Immunol. Methods. 13: 157-167 (1998).-   133. Wang, X. M. et al. A new microcellular cytotoxicity test based    on calcein AM release. Hum. Immunol. 37: 264-270 (1993).-   134. Testa, J. R. & Giordano, A. SV40 and cell cycle perturbations    in malignant mesothelioma. Semin Cancer Biol. 11: 31-8. (2001).-   135. Bosch, F. X. et al. Prevalence of human papillomavirus in    cervical cancer: a worldwide perspective. International biological    study on cervical cancer (IBSCC)-   Study Group. J Natl Cancer Inst. 87: 796-802 (1995).-   136. Elenbaas, B. et al. Human breast cancer cells generated by    oncogenic transformation of primary mammary epithelial cells. Genes    Dev. 15: 50-65. (2001).-   137. Perez-Stable, C., Altman, N. H., Mehta, P. P., Deftos, L. J. &    Roos, B. A. Prostate cancer progression, metastasis, and gene    expression in transgenic mice. Cancer Res. 57: 900-6. (1997).-   138. Rich, J. N. et al. A genetically tractable model of human    glioma formation. Cancer Res. 61: 3556-60. (2001).-   139. Chan, Y. M., Wu, W., Yip, H. K., So, K. F. & Oppenheim, R. W.    Caspase inhibitors promote the survival of avulsed spinal    motoneurons in neonatal rats. Neuroreport. 12: 541-545 (2001).-   140. Makin, G. Targeting apoptosis in cancer chemotherapy. Expert    Opin Ther Targets. 6: 73-84. (2002).-   141. Ho, S. H., Das Gupta, U. & Rieske, J. S. Detection of    antimycin-binding subunits of complex III by photoaffinity-labeling    with an azido derivative of antimycin. J Bioenerg Biomembr. 17:    269-82 (1985).-   142. G, V. O. N. J. & Bohrer, C. Inhibition of electron transfer    from ferrocytochrome b to ubiquinone, cytochrome c1 and duroquinone    by antimycin. Biochim Biophys Acta. 387: 409-24 (1975).-   143. Huang, P., Feng, L., Oldham, E. A., Keating, M. J. &    Plunkett, W. Superoxide dismutase as a target for the selective    killing of cancer cells. Nature. 407: 390-5 (2000).-   144. Wood, L. et al. Inhibition of superoxide dismutase by    2-methoxyoestradiol analogues and oestrogen derivatives:    structure-activity relationships. Anticancer Drug Des. 16: 209-15    (2001).-   145. Gilad, E., Cuzzocrea, S., Zingarelli, B., Salzman, A. L. &    Szabo, C. Melatonin is a scavenger of peroxynitrite. Life Sci. 60:    PL169-74 (1997).-   146. Plattner, R. et al. Differential contribution of the ERK and    JNK mitogen-activated protein kinase cascades to Ras transformation    of HT1080 fibrosarcoma and DLD-1 colon carcinoma cells. Oncogene.    18: 1807-17 (1999).-   147. Davies, H. et al. Mutations of the BRAF gene in human cancer.    Nature. 417: 949-54 (2002).-   148. Patton, S. E. et al. Activation of the ras-mitogen-activated    protein kinase pathway and phosphorylation of ets-2 at position    threonine 72 in human ovarian cancer cell lines. Cancer Res. 58:    2253-9 (1998).-   149. Beck, W. T. & Danks, M. K. Mechanisms of resistance to drugs    that inhibit DNA topoisomerases. Semin Cancer Biol. 2: 235-44    (1991).-   150. Shimizu, S., Konishi, A., Kodama, T. & Tsujimoto, Y. BH4 domain    of antiapoptotic Bcl-2 family members closes voltage-dependent anion    channel and inhibits apoptotic mitochondrial changes and cell death.    Proc Natl Acad Sci USA. 97: 3100-5 (2000).-   151. Rahmani, Z., Huh, K. W., Lasher, R. & Siddiqui, A. Hepatitis B    virus X protein colocalizes to mitochondria with a human    voltage-dependent anion channel, HVDAC3, and alters its    transmembrane potential. J Virol. 74: 2840-6 (2000).-   152. Koppel, D. A. et al. Bacterial expression and characterization    of the mitochondrial outer membrane channel. Effects of n-terminal    modifications. J Biol Chem. 273: 13794-800 (1998).-   153. Xu, X., Decker, W., Sampson, M. J., Craigen, W. J. &    Colombini, M. Mouse VDAC isoforms expressed in yeast: channel    properties and their roles in mitochondrial outer membrane    permeability. J Membr Biol. 170: 89-102 (1999).-   154. Navratilova, I., Sodroski, J. & Myszka, D. G. Solubilization,    stabilization, and purification of chemokine receptors using    biosensor technology. Anal Biochem. 339: 271-81 (2005).-   155. Stenlund, P., Babcock, G. J., Sodroski, J. & Myszka, D. G.    Capture and reconstitution of G protein-coupled receptors on a    biosensor surface. Anal Biochem. 316: 243-50 (2003).-   156. Cliff, M. J., Gutierrez, A. & Ladbury, J. E. A survey of the    year 2003 literature on applications of isothermal titration    calorimetry. J Mol Recognit. 17: 513-23 (2004).-   157. Leavitt, S. & Freire, E. Direct measurement of protein binding    energetics by isothermal titration calorimetry. Curr Opin Struct    Biol. 11: 560-6 (2001).-   158. Rostovtseva, T. & Colombini, M. VDAC channels mediate and gate    the flow of ATP: implications for the regulation of mitochondrial    function. Biophys J. 72: 1954-62 (1997).-   159. Montal, M. & Mueller, P. Formation of bimolecular membranes    from lipid monolayers and a study of their electrical properties.    Proc Natl Acad Sci USA. 69: 3561-6 (1972).-   160. Stewart, S. A. et al. Lentivirus-delivered stable gene    silencing by RNAi in primary cells. Rna. 9: 493-501 (2003).-   161. Lee, A. C., Zizi, M. & Colombini, M. Beta-NADH decreases the    permeability of the mitochondrial outer membrane to ADP by a factor    of 6. J Biol Chem. 269: 30974-80 (1994).-   162. Zizi, M., Forte, M., Blachly-Dyson, E. & Colombini, M. NADH    regulates the gating of VDAC, the mitochondrial outer membrane    channel. J Biol Chem. 269: 1614-6 (1994).-   163. Mannella, C. A. Conformational changes in the mitochondrial    channel protein, VDAC, and their functional implications. J Struct    Biol. 121: 207-18 (1998).-   164. Giepmans, B. N., Adams, S. R., Ellisman, M. H. & Tsien, R. Y.    The fluorescent toolbox for assessing protein location and function.    Science. 312: 217-24 (2006).-   165. Mannella, C. A. Structural analysis of mitochondrial pores.    Experientia. 46: 137-45 (1990).-   166. Peng, S., Blachly-Dyson, E., Forte, M. & Colombini, M. Large    scale rearrangement of protein domains is associated with voltage    gating of the VDAC channel. Biophys J. 62: 123-31; discussion 131-5    (1992).-   167. Peng, S., Blachly-Dyson, E., Colombini, M. & Forte, M.    Determination of the number of polypeptide subunits in a functional    VDAC channel from Saccharomyces cerevisiae. J Bioenerg Biomembr. 24:    27-31 (1992).-   168. Johnson, G. L. & Lapadat, R. Mitogen-activated protein kinase    pathways mediated by ERK, JNK, and p38 protein kinases. Science.    298: 1911-2 (2002).-   169. Inoue, K. et al. Isolation and characterization of    mitochondrial DNA-less lines from various mammalian cell lines by    application of an anticancer drug, ditercalinium. Biochem Biophys    Res Commun. 239: 257-60 (1997).-   170. Inoue, K. et al. Isolation of mitochondrial DNA-less mouse cell    lines and their application for trapping mouse synaptosomal    mitochondrial DNA with deletion mutations. J Biol Chem. 272: 15510-5    (1997).-   171. Chrzanowska-Lightowlers, Z. M., Turnbull, D. M. &    Lightowlers, R. N. A microtiter plate assay for cytochrome c oxidase    in permeabilized whole cells. Anal Biochem. 214: 45-9 (1993).-   172. Schriner, S. E. et al. Extension of murine life span by    overexpression of catalase targeted to mitochondria. Science. 308:    1909-11 (2005).-   173. Levi, S. & Arosio, P. Mitochondrial ferritin. Int J Biochem    Cell Biol. 36: 1887-9 (2004).-   174. He, H. et al. Identification of potent water soluble    purine-scaffold inhibitors of the heat shock protein 90. J Med Chem.    49: 381-90 (2006).-   175. Fury, M. G. et al. A phase I clinical pharmacologic study of    pralatrexate in combination with probenecid in adults with advanced    solid tumors. Cancer Chemother Pharmacol. 57: 671-7 (2006).-   176. Frost, J. A. et al. Simian virus 40 small t antigen cooperates    with mitogen-activated kinases to stimulate AP-1 activity. Mol Cell    Biol 14, 6244-52 (1994).-   177. Cheng, E. H., Sheiko, T. V., Fisher, J. K., Craigen, W. J. &    Korsmeyer, S. J. VDAC2 inhibits BAK activation and mitochondrial    apoptosis. Science 301, 513-7 (2003).-   178. Poyurovsky, M. V. et al. Nucleotide binding by the Mdm2 RING    domain facilitates Arf-independent Mdm2 nucleolar localization. Mol    Cell 12, 875-87 (2003).-   179. Koppel, D. A. et al. Bacterial expression and characterization    of the mitochondrial outer membrane channel. Effects of n-terminal    modifications. J Biol Chem 273, 13794-800 (1998).-   180. Zhen, Y. et al. Development of an LC-MALDI method for the    analysis of protein complexes. J Am Soc Mass Spectrom 15, 803-22    (2004).-   181. Sage, J., Miller, A. L., Perez-Mancera, P. A., Wysocki, J. M. &    Jacks, T. Acute mutation of retinoblastoma gene function is    sufficient for cell cycle re-entry. Nature 424, 223-8 (2003).

Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

1. A compound having formula I:

wherein R₁ is a hydrophobic or hydrophilic substituent, which isattached to one or more positions of at least one carbon atom of thering; R₂ is selected from is selected from H, C₁₋₈alkyl, C₁₋₈alkoxy, 3-to 8-membered carbocyclic or heterocyclic, aryl, heteroaryl,C₁₋₄aralkyl, residues of glycolic acid, ethylene glycol/propylene glycolcopolymers, carboxylate, ester, amide, carbohydrate, amino acid,alditol, OC(R₆)₂COOH, SC(R₆)₂COOH, NHCHR₆COOH, COR₇, CO₂R₇, sulfate,sulfonamide, sulfoxide, sulfonate, sulfone, thioalkyl, thioester,propylphthalimide, and thioether; R₃ is a C₂₋₈ alkoxy; R₆ is selectedfrom H, C₁₋₈alkyl, carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, and alkylheterocycle, wherein each alkyl, carbocycle,aryl, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl, andalkylheterocycle may be optionally substituted with at least onesubstituent; R₇ is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,aryl, carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, alkylheterocycle, and heteroaromatic may be optionallysubstituted with at least one substituent; or an enantiomer, opticalisomer, diastereomer, N-oxide, crystalline form, hydrate, orpharmaceutically acceptable salt thereof.
 2. The compound according toclaim 1, wherein the hydrophilic substituent is selected from the groupconsisting of alcohols, amines, nitro, carboxylic acids, carboxylates,hydroxy, amides, sulfamides, sulfonic acids, sulfonates, sulfates,esters, thiol esters, ethers, thiols, thiolates, thiol ethers,morpholino, fluoroaromatics, piperazines, piperadines, phosphonates, andsalts thereof, and combinations thereof.
 3. The compound according toclaim 2, wherein the hydrophilic substituent is selected from the groupconsisting of NH₂, NO₂, NCOCH₃, and combinations thereof.
 4. Thecompound according to claim 1, wherein R₂ is H or CH₃.
 5. The compoundaccording to claim 1, wherein R₃ is ethoxy or isopropoxy.
 6. A compoundhaving formula Ia:

wherein R₁ is a hydrophobic or hydrophilic substituent, which isattached to one or more positions of at least one carbon atom of thering; R₃ is a C₂₋₈ alkoxy; or an enantiomer, optical isomer,diastereomer, N-oxide, crystalline form, hydrate, or pharmaceuticallyacceptable salt thereof.
 7. The compound according to claim 6, whereinthe hydrophilic substituent is selected from the group consisting ofalcohols, amines, nitro, carboxylic acids, carboxylates, hydroxy,amides, sulfamides, sulfonic acids, sulfonates, sulfates, esters, thiolesters, ethers, thiols, thiolates, thiol ethers, morpholino,fluoroaromatics, piperazines, piperadines, phosphonates, and saltsthereof, and combinations thereof.
 8. The compound according to claim 7,wherein the hydrophilic substituent is selected from the groupconsisting of NH₂, NO₂, NCOCH₃, and combinations thereof.
 9. Thecompound according to claim 6, wherein R₃ is ethoxy or isopropoxy. 10.The compound according to claim 1, which is selected from the groupconsisting of

an enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.
 11. A compound offormula II:

wherein A is selected from the group consisting of C, N, and O; R₁ is ahydrophobic or hydrophilic substituent, which is attached to one or morepositions of at least one carbon atom of the ring with the proviso thatwhen A is C, R₁ is not NH₂ or NO₂; R₂ is selected from H, C₁₋₈alkyl,C₁₋₈alkoxy, 3- to 8-membered carbocyclic or heterocyclic, aryl,heteroaryl, C₁₋₄aralkyl, residues of glycolic acid, ethyleneglycol/propylene glycol copolymers, carboxylate, ester, amide,carbohydrate, amino acid, alditol, OC(R₆)₂COOH, SC(R₆)₂COOH, NHCHR₆COOH,COR₇, CO₂R₇, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,thioalkyl, thioester, propylphthalimide, and thioether; R₃ is a C₂₋₈alkoxy; R₄ is a hydrophilic substituent, which is attached to at leastone position of A, except that when A is O, R₃ is nothing; R₆ isselected from H, C₁₋₈alkyl, carbocycle, aryl, heteroaryl, heterocycle,alkylaryl, alkylheteroaryl, and alkylheterocycle, wherein each alkyl,carbocycle, aryl, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,and alkylheterocycle may be optionally substituted with at least onesubstituent; R₇ is selected from H, C₁₋₈alkyl, C₁₋₈alkenyl, C₁₋₈alkynyl,aryl, carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,alkylheteroaryl, alkylheterocycle, and heteroaromatic may be optionallysubstituted with at least one substituent; or an enantiomer, opticalisomer, diastereomer, N-oxide, crystalline form, hydrate, orpharmaceutically acceptable salt thereof.
 12. The compound according toclaim 11, wherein the hydrophilic substituent is selected from the groupconsisting of alcohols, amines, nitro, carboxylic acids, carboxylates,hydroxy, amides, sulfamides, sulfonic acids, sulfonates, sulfates,esters, thiol esters, ethers, thiols, thiolates, thiol ethers,morpholino, fluoroaromatics, piperazines, piperadines, phosphonates, andsalts thereof, and combinations thereof.
 13. The compound according toclaim 12, wherein the hydrophilic substituent is NCOCH₃.
 14. Thecompound according to claim 11, wherein R₂ is H or CH₃.
 15. The compoundaccording to claim 11, wherein R₃ is ethoxy or isopropoxy.
 16. Thecompound according to claim 11, wherein A is C.
 17. A compound selectedfrom the group consisting of formula IIa, IIb, and IIc:

wherein R₁ is a hydrophobic or hydrophilic substituent, which isattached to one or more positions of at least one carbon atom of thering with the proviso that in formula IIa, R₁ is not NH₂ or NO₂; R₂ isselected from H, C₁₋₈alkyl; R₃ is a C₂₋₈ alkoxy; R₄ and R₅, whenpresent, are independently selected from the group consisting of H andan hydrophilic substituent; or an enantiomer, optical isomer,diastereomer, N-oxide, crystalline form, hydrate, or pharmaceuticallyacceptable salt thereof.
 18. The compound according to claim 17, whichis selected from the group consisting of

an enantiomer, optical isomer, diastereomer, N-oxide, crystalline form,hydrate, or pharmaceutically acceptable salt thereof.
 19. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound according to any one of claims 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or
 18. 20. A method of treatinga condition in a mammal, comprising administering to the mammal atherapeutically effective amount of a compound according to any one ofclaims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.21. The method according to claim 20, wherein the mammal is a human. 22.The method according to claim 20, wherein the condition is cancer. 23.The method according to claim 22, wherein the cancer is selected fromthe group consisting of leukemia, non-small cell lung carcinoma, coloncancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostatecancer, breast cancer, and pancreatic cancer.
 24. The method accordingto claim 20, further comprising conjointly administering to the mammalan agent that kills cells through an apoptotic mechanism.
 25. Themethod, according to claim 24, wherein the agent is a chemotherapeuticagent.
 26. The method according to claim 25, wherein thechemotherapeutic agent is selected from the group consisting of anEGF-receptor antagonist, arsenic sulfide, adriamycin, cisplatin,carboplatin, cimetidine, caminomycin, mechlorethamine hydrochloride,pentamethylmelamine, thiotepa, teniposide, cyclophosphamide,chlorambucil, demethoxyhypocrellin A, melphalan, ifosfamide,trofosfamide, Treosulfan, podophyllotoxin or podophyllotoxinderivatives, etoposide phosphate, teniposide, etoposide, leurosidine,leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol,megestrol, methopterin, mitomycin C, ecteinascidin 743, busulfan,carmustine (BCNU), lomustine (CCNU), lovastatin,1-methyl-4-phenylpyridinium ion, semustine, staurosporine, streptozocin,phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate,thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin,cytarabine (ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine,doxorubicin hydrochloride, leucovorin, mycophenolic acid, daunorubicin,deferoxamine, floxuridine, doxifluridine, raltitrexed, idarubicin,epirubican, pirarubican, zorubicin, mitoxantrone, bleomycin sulfate,actinomycin D, safracins, saframycins, quinocarcins, discodermolides,vincristine, vinblastine, vinorelbine tartrate, vertoporfin, paclitaxel,tamoxifen, raloxifene, tiazofuran, thioguanine, ribavirin, EICAR,estramustine, estramustine phosphate sodium, flutamide, bicalutamide,buserelin, leuprolide, pteridines, enediynes, levamisole, aflacon,interferon, interleukins, aldesleukin, filgrastim, sargramostim,rituximab, BCG, tretinoin, betamethosone, gemcitabine hydrochloride,verapamil, VP-16, altretamine, thapsigargin, oxaliplatin, iproplatin,tetraplatin, lobaplatin, DCP, PLD-147, JM118, JM216, JM335, satraplatin,docetaxel, deoxygenated paclitaxel, TL-139, 5′-nor-anhydrovinblastine(hereinafter: 5′-nor-vinblastine), camptothecin, irinotecan (Camptosar,CPT-11), topotecan (Hycamptin), BAY 38-3441, 9-nitrocamptothecin(Orethecin, rubitecan), exatecan (DX-8951), lurtotecan (GI-147211C),gimatecan, homocamptothecins diflomotecan (BN-80915) and9-aminocamptothecin (IDEC-13′), SN-38, ST1481, karanitecin (BNP1350),indolocarbazoles (e.g., NB-506), protoberberines, intoplicines,idenoisoquinolones, benzo-phenazines, NB-506, and combinations thereof.27. A method of treating a condition in a mammal, comprisingadministering to the mammal a therapeutically effective amount of apharmaceutical composition according to claim
 20. 28. The methodaccording to claim 27, wherein the mammal is a human.
 29. The methodaccording to claim 28, wherein the condition is cancer.
 30. The methodaccording to claim 29, wherein the cancer is selected from the groupconsisting of leukemia, non-small cell lung carcinoma, colon cancer, CNScancer, melanoma, ovarian cancer, renal cancer, prostate cancer, breastcancer, and pancreatic cancer.
 31. The method according to claim 27,further comprising conjointly administering to the mammal an agent thatkills cells through an apoptotic mechanism.
 32. The method according toclaim 31, wherein the agent is a chemotherapeutic agent.
 33. The methodaccording to claim 32, wherein the chemotherapeutic agent is selectedfrom the group consisting of an EGF-receptor antagonist, arsenicsulfide, adriamycin, cisplatin, carboplatin, cimetidine, caminomycin,mechlorethamine hydrochloride, pentamethylmelamine, thiotepa,teniposide, cyclophosphamide, chlorambucil, demethoxyhypocrellin A,melphalan, ifosfamide, trofosfamide, Treosulfan, podophyllotoxin orpodophyllotoxin derivatives, etoposide phosphate, teniposide, etoposide,leurosidine, leurosine, vindesine, 9-aminocamptothecin,camptoirinotecan, crisnatol, megestrol, methopterin, mitomycin C,ecteinascidin 743, busulfan, carmustine (BCNU), lomustine (CCNU),lovastatin, 1-methyl-4-phenylpyridinium ion, semustine, staurosporine,streptozocin, phthalocyanine, dacarbazine, aminopterin, methotrexate,trimetrexate, thioguanine, mercaptopurine, fludarabine, pentastatin,cladribin, cytarabine (ara C), porfiromycin, 5-fluorouracil,6-mercaptopurine, doxorubicin hydrochloride, leucovorin, mycophenolicacid, daunorubicin, deferoxamine, floxuridine, doxifluridine,raltitrexed, idarubicin, epirubican, pirarubican, zorubicin,mitoxantrone, bleomycin sulfate, actinomycin D, safracins, saframycins,quinocarcins, discodermolides, vincristine, vinblastine, vinorelbinetartrate, vertoporfin, paclitaxel, tamoxifen, raloxifene, tiazofuran,thioguanine, ribavirin, EICAR, estramustine, estramustine phosphatesodium, flutamide, bicalutamide, buserelin, leuprolide, pteridines,enediynes, levamisole, aflacon, interferon, interleukins, aldesleukin,filgrastim, sargramostim, rituximab, BCG, tretinoin, betamethosone,gemcitabine hydrochloride, verapamil, VP-16, altretamine, thapsigargin,oxaliplatin, iproplatin, tetraplatin, lobaplatin, DCP, PLD-147, JM118,JM216, JM335, satraplatin, docetaxel, deoxygenated paclitaxel, TL-139,5′-nor-anhydrovinblastine (hereinafter: 5′-nor-vinblastine),camptothecin, irinotecan (Camptosar, CPT-11), topotecan (Hycamptin), BAY38-3441, 9-nitrocamptothecin (Orethecin, rubitecan), exatecan (DX-8951),lurtotecan (GI-147211C), gimatecan, homocamptothecins diflomotecan(BN-80915) and 9-aminocamptothecin (IDEC-13′), SN-38, ST1481,karanitecin (BNP1350), indolocarbazoles (e.g., NB-506), protoberberines,intoplicines, idenoisoquinolones, benzo-phenazines, NB-506, andcombinations thereof.
 34. A compound selected from the group consistingof

or an enantiomer, optical isomer, diastereomer, N-oxide, crystallineform, hydrate, or pharmaceutically acceptable salt thereof.
 35. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound according to claim
 34. 36. A method of treating acondition in a mammal, comprising administering to the mammal atherapeutically effective amount of a compound according to claim 34.37. The method according to claim 36, wherein the mammal is a human. 38.The method according to claim 36, wherein the condition is cancer. 39.The method according to claim 38, wherein the cancer is selected fromthe group consisting of leukemia, non-small cell lung carcinoma, coloncancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostatecancer, breast cancer, and pancreatic cancer.
 40. The method accordingto claim 36, further comprising conjointly administering to the mammalan agent that kills cells through an apoptotic mechanism.
 41. The methodaccording to claim 40, wherein the agent is a chemotherapeutic agent.42. The method according to claim 41, wherein the chemotherapeutic agentis selected from the group consisting of an EGF-receptor antagonist,arsenic sulfide, adriamycin, cisplatin, carboplatin, cimetidine,caminomycin, mechlorethamine hydrochloride, pentamethylmelamine,thiotepa, teniposide, cyclophosphamide, chlorambucil,demethoxyhypocrellin A, melphalan, ifosfamide, trofosfamide, Treosulfan,podophyllotoxin or podophyllotoxin derivatives, etoposide phosphate,teniposide, etoposide, leurosidine, leurosine, vindesine,9-aminocamptothecin, camptoirinotecan, crisnatol, megestrol,methopterin, mitomycin C, ecteinascidin 743, busulfan, carmustine(BCNU), lomustine (CCNU), lovastatin, 1-methyl-4-phenylpyridinium ion,semustine, staurosporine, streptozocin, phthalocyanine, dacarbazine,aminopterin, methotrexate, trimetrexate, thioguanine, mercaptopurine,fludarabine, pentastatin, cladribin, cytarabine (ara C), porfiromycin,5-fluorouracil, 6-mercaptopurine, doxorubicin hydrochloride, leucovorin,mycophenolic acid, daunorubicin, deferoxamine, floxuridine,doxifluridine, raltitrexed, idarubicin, epirubican, pirarubican,zorubicin, mitoxantrone, bleomycin sulfate, actinomycin D, safracins,saframycins, quinocarcins, discodermolides, vincristine, vinblastine,vinorelbine tartrate, vertoporfin, paclitaxel, tamoxifen, raloxifene,tiazofuran, thioguanine, ribavirin, EICAR, estramustine, estramustinephosphate sodium, flutamide, bicalutamide, buserelin, leuprolide,pteridines, enediynes, levamisole, aflacon, interferon, interleukins,aldesleukin, filgrastim, sargramostim, rituximab, BCG, tretinoin,betamethosone, gemcitabine hydrochloride, verapamil, VP-16, altretamine,thapsigargin, oxaliplatin, iproplatin, tetraplatin, lobaplatin, DCP,PLD-147, JM118, JM216, JM335, satraplatin, docetaxel, deoxygenatedpaclitaxel, TL-139, 5′-nor-anhydrovinblastine (hereinafter:5′-nor-vinblastine), camptothecin, irinotecan (Camptosar, CPT-11),topotecan (Hycamptin), BAY 38-3441, 9-nitrocamptothecin (Orethecin,rubitecan), exatecan (DX-8951), lurtotecan (GI-147211C), gimatecan,homocamptothecins diflomotecan (BN-80915) and 9-aminocamptothecin(IDEC-13′), SN-38, ST1481, karanitecin (BNP1350), indolocarbazoles(e.g., NB-506), protoberberines, intoplicines, idenoisoquinolones,benzo-phenazines, NB-506, and combinations thereof.
 43. A method oftreating a condition in a mammal comprising administering to the mammala therapeutically effective amount of a pharmaceutical compositionaccording to claim
 35. 44. The method according to claim 43, wherein themammal is a human.
 45. The method according to claim 43, wherein thecondition is cancer.
 46. The method according to claim 45, wherein thecancer is selected from the group consisting of leukemia, non-small celllung carcinoma, colon cancer, CNS cancer, melanoma, ovarian cancer,renal cancer, prostate cancer, breast cancer, and pancreatic cancer. 47.The method according to claim 43, further comprising conjointlyadministering to the mammal an agent that kills cells through anapoptotic mechanism.
 48. The method according to claim 47, wherein theagent is a chemotherapeutic agent.
 49. The method according to claim 48,wherein the chemotherapeutic agent is selected from the group consistingof an EGF-receptor antagonist, arsenic sulfide, adriamycin, cisplatin,carboplatin, cimetidine, caminomycin, mechlorethamine hydrochloride,pentamethylmelamine, thiotepa, teniposide, cyclophosphamide,chlorambucil, demethoxyhypocrellin A, melphalan, ifosfamide,trofosfamide, Treosulfan, podophyllotoxin or podophyllotoxinderivatives, etoposide phosphate, teniposide, etoposide, leurosidine,leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol,megestrol, methopterin, mitomycin C, ecteinascidin 743, busulfan,carmustine (BCNU), lomustine (CCNU), lovastatin,1-methyl-4-phenylpyridinium ion, semustine, staurosporine, streptozocin,phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate,thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin,cytarabine (ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine,doxorubicin hydrochloride, leucovorin, mycophenolic acid, daunorubicin,deferoxamine, floxuridine, doxifluridine, raltitrexed, idarubicin,epirubican, pirarubican, zorubicin, mitoxantrone, bleomycin sulfate,actinomycin D, safracins, saframycins, quinocarcins, discodermolides,vincristine, vinblastine, vinorelbine tartrate, vertoporfin, paclitaxel,tamoxifen, raloxifene, tiazofuran, thioguanine, ribavirin, EICAR,estramustine, estramustine phosphate sodium, flutamide, bicalutamide,buserelin, leuprolide, pteridines, enediynes, levamisole, aflacon,interferon, interleukins, aldesleukin, filgrastim, sargramostim,rituximab, BCG, tretinoin, betamethosone, gemcitabine hydrochloride,verapamil, VP-16, altretamine, thapsigargin, oxaliplatin, iproplatin,tetraplatin, lobaplatin, DCP, PLD-147, JM118, JM216, JM335, satraplatin,docetaxel, deoxygenated paclitaxel, TL-139, 5′-nor-anhydrovinblastine(hereinafter: 5′-nor-vinblastine), camptothecin, irinotecan (Camptosar,CPT-11), topotecan (Hycamptin), BAY 38-3441, 9-nitrocamptothecin(Orethecin, rubitecan), exatecan (DX-8951), lurtotecan (GI-147211C),gimatecan, homocamptothecins diflomotecan (BN-80915) and9-aminocamptothecin (IDEC-13′), SN-38, ST1481, karanitecin (BNP1350),indolocarbazoles (e.g., NB-506), protoberberines, intoplicines,idenoisoquinolones, benzo-phenazines, NB-506, and combinations thereof.